MCB 2000: Lab Practical

→ What is SDS page used for?

- Determine protein size - Identify protein - Quantify protein - Starting point for western blots

pKa =

-logKa

pH =

-log[H+]

What does the Henderson Hasselbach equation allow us to related?

A way for you to relate pH and pKa to your concentrations of weak acid and conjugate base.

What is a free radical?

An atom containing at least on unpaired electron in its outermost shell

Distribution coefficient (Kw)

Distribution Coefficient (Kw): Use elution volume, void volume and total volume.

Lab 2: How are amino acids grouped?

Groupings are based on the side chains: → Polar side chains: → Non polar side chains: → Acidic and basic side chains: Both acidic and basic are electrically charged

What does a titratable R group mean?

In the purple box, these amino acids have a titratable R group; this means they are able to lose a proton or gain a proton.

How do you calculate the pI of alanine?

This pI is important because in a titration curve it is going to tell us where our moles of base equal our moles of acid. (PI is important because we know that we have equal moles of our base and acid) - Image in notes

What type of Ka and pKa do weak acids have? Strong acids?

Weak acids have a small Ka, but a large pKa. Strong acids have large Kas, but small pKas.

Noncompetitive inhibition

inhibitor binds elsewhere on the enzyme; alters active site so that the substrate cannot bind

When [A-] = [HA] then

pH = pKa pKa is the pH at which [A-] = [HA]

What is pH

pH is actually the negative log of our H+ concentration

Define transmittance

→ Transmittance (T): It/Io (Transmittance is comparing the ratio between the light that's made it through our sample over the original light we started with.)

What is an antioxidant?

- A substance the delays or prevents oxidation of a substrate

Alternatives to pierce 660 nm - most affected by surfactants

- Bradford assay : colorimetric assay or protein dye binding - Biuret assay: Colorimetric assay or copper interaction with peptide bonds - Absorbance at 280 nm (OD280) --> Requires additional information (extinction coefficient) --> dependent on tyrosine, tryptophan, phenylalanine residues --> affected by sample impurities - absorbance at 215 and 225 nm -- where peptide bonds absorb

Pierce 660 nm Assay: Protein quantification

- Dye metal complex based colorimetric assay --> concentration of protein related to color change monitored by spectrophotometry - used for fast and accurate and more reliable protein concentration measurements - Protein concentration range of 25-2000 ug/mL - Presence of detergents/reducing agents does not interfere with the assay. - Low volume of sample.

Classification of antioxidants

- Enzymatic (antioxidant svavenging enzymes) --> Superoxide dismutase --> Catalase --> Glutathionine peroxidase and reductase - Non enzymatic (free radical scavengers) --> Endogenous: Uric acid, Melatonin --> Exogenous: Vitamin E, Vitamin C, Carotenoids

elution of bound LDH

- High concentration of imidazole in the buffers competes with His residues to bind nickel ions in stationary phase - Imidazole replaces the bound protein by binding to the column itself Imidazole will bind to the nickel residues and because of the competition, our LDH will be eluted out. Imidazole is replacing bound protein by binding to the column itself.

What does the pierce 660 nm assay use?

- The assay uses polyhydroxybenzenesulfonephthalein-type dye and a transition metal complex based total protein determination method - the dye meal complex has a reddish brown color that changes to green on binding to protein - There is a shift in the absorption maximum of the dye metal complex fro 450 to 660 nm. The dye metal complex has a reddish brown color; when protein is added the color changes to green, shifting the absorption maximum We can monitor how much of our assay is moving from 450 to 660 depending on how much protein is in our sample We're looking for this color change from red to green which is going to indicate how much protein is actually in there. The more green, the more protein in the sample.

ROS can be damaging to the cell - explain

- oxidative stress: when the amount of ROS produced exceeds the removal by cellular defense mechanisms - Leads to damaged lipids, DNA/nucleic acids, and proteins - linked to Alzheimer's disease, cancer, heart disease, Parkinson disease, and others If our body can't deal with these reactive oxygen species quickly enough or they get overwhelmed,they can really be dangerous to the cell.

What are other factors that affect buffering capacity?

--> The volume of our buffer (greater volume means more acid or base your able to add into a buffer that will be neutralized as opposed to if we have a small amount of that buffer → Concentrations of our weak acid and conjugate base.

Lab 2: What two methods will you use to determine the identity of your unknown amino acid?

1. Titration 2. Thin Layer Chromatography (TLC)

pH + pOH = ?

14

Describe the basic structure of amino acids

4 different parts: Amine group Alpha carbon: where all these groups come off of Carboxyl group R group: Each different amino acid will have a different R group

What is an absorption spectrum?

A graph plotting a pigment's light absorption versus wavelength Absorption spectrum: Every substance has a unique absorption spectrum; will absorb light in different ways at different wavelengths. So there's going to be a lambda max

What is SDS-page?

A negative denaturing gel that is used to determine molecular weight or size → Sodium Dodecyl Sulfate Polyacrylamide Gel electrophoresis A method of separating denatured proteins based on their size By running SDS, well get a better size of the molecular weights in each of our peak fractions

Describe what a small Km means and what a large Km means

A small Km means our ES complex is pretty stable. A pretty large Km means a less stable ES complex.

How to find out unknown 2 concentrations since we cant extrapolate our curve.

Absorbance of unknown 2 = 1.8; off our graph Take unknown 2, and make a dilution. Make a 2 fold dilution of our unknown 2. This dilutes our unknown and gives us an absorbance that will fall on our standard curve. We would have a new solution of unknown 2, a diluted solution. If we made our 3 fold dilution, we get an absorbance of 0.7. Our new absorbance of our unknown is on our standard curve. This gives us a concentration of 14 micromolar. The way we determine our actual concentration of unknown 2 is to multiply by the dilution we just made. Multiply concentration of 14 by 3 (since we made a 3 fold dilution). So we get 42 micromolar for the concentration of unknown 2. What would you do if a 2-fold dilution still gives an absorbance reading that does not fall on the standard curve? → You have to go back and make a new dilution of the unknown solution. What about unknown 3 absorbance =0.1? Can you find the concentration? → No. The lowest value we have for our standard curve is 0.2. This is out of the range of our standard curve.

Define accuracy

Accuracy: Closeness of experimental values to the theoretical value; percent error

Define affinity chromatography

Affinity chromatography (stationary phase: Biopecific resin - ligand binding).

Describe Alanine as we increase pH as a form of understanding a titration curve

Alanine does not have a titratable R group. In tis most acidic form, you will have the first form of alanine, meaning everything is going to be protonated. Neutral charge on our carboxyl group (cause we have a proton there) and we'll have a positive charge on our amine group because we have this NH3+ here. As you increase the pH, you reach your pK1. At pK1 we have equal amounts of our conjugate base and weak acid, which means we're going to have equal amounts of the first two molecules (on the left). Because we have equal amounts of our conjugate base and weak acid, we have a really good buffer at pK1, seen by the plateauing. As we increase our pH, we see those buffering effects at pK2. We're losing the proton on our amine group. At pK2 we have equal amounts of our conjugate base and weak acid.

What are amino acids?

Amino acids are the building blocks for our protein structure; come together through a dehydration reaction to form peptide bonds

IF a sample rich in antioxidants is added to the above assay, would you expect the absorbance of NBT at 560 nm to increase or decrease?

Answer to question: We should expect it to decrease because if we add in a sample rich in antioxidants to this assay, we are not going to get so much formation of our superoxide. And without formation of our superoxide, we are not going to get so much of the NBT being reduced and forming that blue color. So when we look at absorbance at 560 nanometers, we're going to see that absorbance decrease.

Cellular Defense Mechanisms:

Antioxidant scavenging enzymes: - superoxide dismutase - catalase - Gluthathione peroxidase Repair processes Degradation of damaged proteins and apoptosis Antioxidant scavenging enzymes help clear our cells of those free radicals. If these repair processes can help the cell enough, the cell can undergo degradation of damaged protein and even apoptosis.

The different wavelengths at which we will be monitoring elution of proteins

As she mentioned we will be monitoring the absorbance of these fractions to determine what species is in each fraction. And we can do this by monitoring these fractions at a number of different wavelengths. So the first wavelength is 280 nm. And this will tell you where your proteins of interest are. We can do this because we see an increase in absorbance of some of our amino acids at 280 nanometers. We're also going to be monitoring at 400 nm. What we should have noticed about cytochrome c at the beginning was that cytochrome c coordinates a heme group. This heme group is going to absorb at 400 nanometers. So where we see a peak absorbance at 400 and also at 280 is going to tell us in what fraction Cytochrome C is present. Ovalbumin doesn't coordinate a heme. So to determine what fraction ovalbumin is located in we'll be monitoring again just at 280 nm. We also want to identify what our void volume is and what our total volume is. To do this, our void volume will be looking for where Dextran Blue comes off of the column. To determine what our total volume is, well be monitoring where vitamin B-12 comes. So for dextram blue will be monitoring our fractions for an absorbance at 640 nanometers. For vitamin B-12 we will be looking for a peak absorbance at 360 nm.

At different pKa's that glycine has:

As we increase from a low pH, our molecule transitions and loses its proton on its carboxyl group. This transition is known as our pKaC. When we transition from the neutrally charged glycine to the negatively charged glycine, the transition happens again and the proton is lost on the amine group. This transition is our pKaN.

What occurs in a basic environment?

Basic environment: High pH. We have a high concentration of OH negative or hydroxyl ions Because now we have a very high concentration of hydroxyl ions, our glycine is going to want to donate its protons. Because our glycine wants to donate these protons, we end up with a slightly different structure. On our carboxyl end, glycine has now donated that proton and we end up with a COO- group. On our amine group, we have donated a proton and end up with an NH2 group.

Define Bronsted base

Bronstead base: H+ or proton acceptor

Define Bronsted acid

Bronsted acid: A substance that is an H+ or proton donor

What is buffering capacity?

Buffering capacity is the moles or acid or base required to change the pH by 1

What's the pI of Glu-Gly Lys? --> Step 1: Definition of PI

By definition of pI, we are determining the pH at which the tripeptide has a net-neutral charge

How do you calculate dilution?

C1V1 = C2V2 - image on notes

Western blot - Color development reaction

Color development reaction: Our BCIP is one of the detection reagents (alkaline phosphatase substrates) and when alkaline phosphatase reacts with this BCIP we get this indoxyl intermediate. Then this indoxyl intermediate interacts with our NBT. This NBT is also in our detection regent and then when these two interact, this is what is creating this indigo precipitate. Finally we are adding our EDTA.

What does the concentration of OH- tell us?

Concentration of OH- tells us about our base concentration in water and is also 1 * 10^-7 M

Elution at 400 nm

Cytochrome C coordinates a heme group and will absorb at 400 nm. So we look at this light blue line and see a peak over the smaller purple peak, indicating we have a protein present at this fraction and also a protein that is coordinating a heme, which would mean that this fraction is cytochrome C. This is how we figure out where our proteins of interest are and what factions they are located at.

What are the different components of a spectrophotometer?

Different components of a spectrophotometer: Start with a lightsource. This lightsource provides light. Light goes through a lens, and this lens focuses the light onto the monochromator. This takes the light and splits it into different wavelengths. Light travels through a wavelength selector so we get rid of the wavelength we don't want and keep just our wavelength of interest. Our wavelength of interest will travel through our sample (in a cuvette). The light that comes from our monochromator is our Io. The light that gets through the sample is known as our It. The It is read by the detector and finally we have a digital display that makes it easy for us to record a number.

How do antioxidants prevent oxidation?

Directly inhibits oxidation reaction by becoming oxidized themselves Trap unstable free radical intermediates

Why is EDTA added to your western blot?

EDTA is going to stop the color development reaction by taking away these metal ions. So it deactivates alkaline phosphatase. Our substrates aren't able to react with AP and we don't get purple color. EDTA is a chelating agent that sequesters metal ions --- deactivates alkaline phosphatase

Elution volume (Ve)

Elution Volume (Ve): Volume of the peak fractions (Fraction that has ovalbumin and one that has cytochrome C).

elution at 360 nm

Finally, we want to look at the total volume of our column using vitamin b-12. This will absorb at 360 nm which is the green line. Vitamin B-12 is small so it will get caught up in these pores and will be the last thing to elute out of our column.

Elution at 640 nm

For the void volume, we look at dextran blue which absorbs at 640 nm. Dextran blue is much larger than our fractionation range which means it's not going to interact with any of the beads or go into any of those pores, it's going to elute right out. So it will be the first peak we're looking for

What is a reactive oxygen species?

Free radicals derived from molecular oxygen Examples: Superoxide anion, peroxide, hydroxyl radical

Define Gel Filtration chromatography

Gel filtration chromatography (Stationary phase: Gel beads with range of pore sizes) which separates proteins based on size.

Describe the conditions at a low pH as it relates to glycine

Glycine has an H group as its side chain, so we are not dealing with a titratable side chain in this case. If we're at a low pH, we're going to be in an acidic environment. This means we have a high concentration of protons (h+). Because we have a high concentration of protons in solution, our glycine is going to want to hold onto its own protons because there's no need for it to donate them into the environment because of this high concentration. This results in this carboxyl group being a COOH (neutral charge) and our amine group being an NH3+ (positive charge) group.

The concentration of H3O+ is...

H+ does not exist freely in water; so instead their represented by hydronium (H3O+) Concentration of H3o+ is the concentration of protons in our solution. Represents the acid concentration of water; for ionization of water this is equal to 1 * 10^-7 M.

How are reactive oxygen species formed?

Here we see our respiratory complexes. These are located in the mitochondria. So at the left we have the matrix, this is the innermost compartment of our mitochondria. We have the inner membrane and the intermembrane space on the left side of the matrix. These respiratory complexes are in charge of creating energy for our cells. But in the process of creating energy they can also create free radicals. And so as you can see here with complex I or complex III, we have these reactive oxygen species being formed

If pH is greater than pKa, does the ratio of conjugate base to weak acid increase or decrease?

If our pH is greater than our pKa it means that we're in a basic environment, which means that we'll have more of our conjugate base than we do our weak acid, so this ratio would increase.

Will the pH of a solution with [H+] > water be greater or less than 7?

If we have a lot of protons in our solution, we're in an acidic environment. This means we have a pH of less than 7, an acidic pH.

Describe the use of differing concentrations of imidazole

In the first column, we have loaded our sample but added no other buffers. So we have our nickel-NTA beads sticking out here, our nickel ions are right there. And at the top we can see that we have a His-tagged LDH. Our his-tagged LDH is interacting with our nickel ion. We also have these blue spheres which are representing different proteins because we're loading in E.coli lysate were going to have a lot of proteins and some of those proteins are going to have histidine residues in them because histidine is an amino acid. So our antive histidine residues, and some of our other proteins, are going to interact with our column. This is the reason that we are going to have to add increasing amounts of imidazole. We next are going to add an intermediate amount of imidazole. What's going to happen when we add an intermediate amount of imidazole is that the imidazole is going to interact with the nickel ions that were interacting with the other random proteins. So when we add this intermediate imidazole in, the random proteins are going to elute out of our column. So we get rid of the unwanted proteins that our stuck to our column When we start adding in higher concentration of imidazole, that's when the imidazole is going to interact with the nickel ions that were interacting with our His-tagged LDH. With These high concentrations of imidazole, we're now having imidazole interact with most of our columns so our protein of interest, our LDH, is now going to start eluting out of the column. So the reason we have to add different concentrations of imidsazole is because we want to make sure that we're actually collecting our LDH protein of interest through our high imidazole concentrations but getting rid of first, any proteins we might not want.

Elution Profile

In this experiment, we are asked to create an elution profile. We'll be collecting a number of different fractions that come off of our column and each of these fractions have a different elution volume. They'll also have different absorbance values for these four wavelengths. So when we make the solution profile, you'll have the elution volume of each of our fractions on the x axis and the absorbance value on the Y-axis.

Define independent conformation

Independent confirmation: Use two different techniques to come to the same conclusion about our question.

Define Ion exchange chromatography

Ion exchange chromatography; exploits different charges of different molecules. Going to separate things based on charge.

What is the isoelectric point?

Isoelectric point is where there is a neutral charge

A standard curve: the do's and the dont's

Issues that should be addressed; - There is a data point not falling on the line. An outlier, for better lack of words. This outlier, for a proper standard curve, should just be taken out. - We only have one set of data plotted. They averaged the two trials of the standards of phenol red together and plotted one value. Don't do this. Keep trials separate.

What is Km?

Km = Our Michaelis Constant. The units of this is going to be molar or a concentration unit. We can see that we can define Km by using our Vmax value. Km is actually the substrate concentration value that correlates to Vmax/2.

How do you calculate the Kw value?

Kw = [H+] * [OH-] = 1 * 10^-14 M

How to create a standard curve:

Label X-axis with independent variable; final concentrations of phenol red (label: Concentration of phenol red in water in micromolar) Label Y-axis with dependent variable; absorbance at 510 nm. Plot known data points. Draw a line of best fit (through as many points as possible) Identify any outliers; ones that don't fit in the best fit line. Circle and label outlier so you show you're not considering it. Figure out concentration of unknown 1. Find absorbance and draw straight over to our line. Draw a line straight down to concentration and fill in the table. Plot unknown 2. Has an absorbance that doesn't fall on standard curve range. So we can't plot this unknown concentration just using this data. What we should do, is make a dilution of unknown 2 and measure the absorbance. Complete both ⅓ dilution and ⅕ dilution. Multiply concentration by our dilution factor. Potential errors: → Extrapolating best fit line; the concentration we calculate isn't going to be accurate → Not measuring standards in duplicate (2 sets of these standards) → Dont average the two absorbance readings → Descriptive title; standard curve of phenol red

Define lambda max

Lambda max: wavelength at which maximum amount of light is absorbed by solution. What is the lambda max of ADP?Look at an absorption spectrum. Specifically, look at where absorbance is maximum for ADP. We notice that max absorbance happens at the highest point of the curve. So our lambda max for ADP would equal 265 nanometers. What about BSA? Focus on the overall maximum.

Describe the use of a spectrophotometer

Measures the fraction of light transmitted through a solution a function of wavelength. Some uses: → Determine protein concentration. → Assay enzymatic activity. → Characterize chemical properties.

Calculate molarity

Molarity (M): Number of moles of solute in exactly one liter of solution

Why should you never extrapolate a standard curve?

Never extrapolate a standard curve. The reason for this is that our standard curve isn't always linear. The area where the graph is linear is where our Beer lambert law holds: A = Ebc. Anywhere outside the linear area, this doesn't hold true. The reason for this is you can have errors in looking at a concentration this low (spectrophotometer might be not sensitive enough) or you can have saturation of color (spectrophotometer can't tell a really concentrated solution from a really really concentrated solution.) Only look at the region of the standard curve that is linear. We only want to look at our standard curve within the range of concentrations of our standards.

What was the protein of interest in the lab?

Ni 2+ binds to his residues which will be the protein of interest today (LDH)

What type of column did we use in the affinity chromatography lab?

Ni-NTA column (stationary phase) used for today's lab: nitriloacetic acid (NTA) chelates (forms coordinate bonds with) Ni2+ ions in the column Nickel NTA beads will be the stationary phase and NTA chelates nickel ions in the column. The column has these beads that use this NTA and coordinate the nickel ions. Nickel ions bind to histidine residues attached to our protein of interest.

What type of amino acids do we have? What are they based off of?

Non polar and polar amino acids Aromatic amino acids Positively and negatively charged amino acids The above three bullets are based on the characteristics of the amino acids R group

Which will travel further up the TLC plate, a nonpolar amino acid or a polar one?

Nonpolar; our mobile phase mixture is nonpolar. Remember, like dissolves like. So a non polar mobile phase will move nonpolar amino acids further up the TLC plate.

Define Normal (N)

Normal (N): Number of reactive equivalents per liter of solution; → Equivalent = weight of a substance that will combine with 1mol H+ or OH- HCl→ H+ + Cl- 1mol H+ can react with 1mol OH- so HCl= 1N

Are the primary and secondary antibodies unique to the different proteins?

Now we have different primary and secondary antibodies for each different membranes we have so the important thing we have to remember today is to make sure we're using the correct primary and secondary antibody on each of our blots.

Describe the different levels of structure in amino acids

Peptide bonds form and you get the primary structure of your amino acid which then turns into a secondary structure where you get helices and beta sheets. These can form tertiary structures. Finally, if different subunits come together, you can get the quaternary structures. If an R group has a titratable side group, this will also have a pKa value

Pierce 660 nm Assay - what does it allow us to do?

Pierce 660 nm Assay: This allows us to quantify our protein samples in another way → Allows for a wide range of protein concentration quantification → If we are in a lab and our sample is critical, it wont use much of our sample up.

Dietary antioxidants

Plant derived polyphenols - Found in fruits, nuts, vegetables, wine, tea, and coffee - Contain readily oxidizable (-OH) substitutions which an act as antioxidants Vitamin E, Vitamin C, carotenoid and mineral selenium These are antioxidants that we can get in our everyday life that can help ourselves balance out these free radicals or reactive oxygen species.

Define precision

Precision: Closeness of experimental values to one another; standard deviation

Western blotting

Procedure that uses labeled antibodies to detect specific antigens in a mixture of proteins separated according to their molecular weight → Allows you to identify proteins of interest with specific antibodies → What antibodies do is recognize antigens. In this case, our antibody is going to specifically recognize your proteins of interest-- so either cytochrome c or ovalbumin. By using ovalbumin antibodies or cytochrome c antibodies, we'll be able to clearly identify where your proteins of interest are and in what fractions there in.

What is a reaction plot?

Reaction plot: Progress of reaction vs. free energy of reaction

How do you calculate Rf value?

Rf = Distance of substance (cm) / distance of solvent (cm); where the solvent front moves. Allows us to compare the controls to our unknown to identify the unknown.

SDS page vs. Western blot

SDS page vs. western blot: Western blot is so important has to do with the results we can get from just staining the SDS page gel. The fourth box that the piece of gel went in had blue stain in it. When we look at our stained gel, we should see something like this, a lot of protein bands picked up by this stain. So its not very clear as to which bands in this stain gel are of our protein of interest. What a western blot is going to allow us to do is to treat our proteins of interest that have been transferred from the gel onto this membrane and these antibodies will highlight only specific bands. SO by completing our western blot, We go from the stain gel to a wetsern blot that is going to only specifically highlight our proteins of interest (right image)

Calculating the pH: if you mixed 50 mL of 0.1 M Tris acid with 60 mL of 0.2M Tris base what will the pH of the buffer be? what is the molarity of the resulting buffer?

See screenshot on computer - Use C1V1=C2V2 to determine the molarity of acid and base. Plug numbers into the HH equation.

By doing this pierce 660 assay...

So by doing this Pierce 660 assay, we're going to get a more accurate reading of our protein concentration but we're also going to be able to compare the concentration values we get from our assay to the concentration values we calculated using our absorbance at 280 nm.

What is the Fab domain?

So we have our antigens and we can think of the antigens of our different proteins of interest. It is what this primary antibody is going to recognize -- So cytochrome c, ovalbumin or beta- galactosidase. The part that is actually going to recognize the antigen and bind to it is called the fab domain.

Describe thin layer chromatography.

Stationary phase is silica gel and mobile phase is an organic solvent. Rate at which different amino acids move depends on H-bond interactions with silica and solubility in solvent. When finished, spray reacts with amino acids to produce purple spots. Retention value of amino acid = distance travelled by component / distance travelled by solvent. Determine your unknown by comparing it to known amino acids (controls) Stationary phase = polar silica gel (crispy piece of paper with silica on it) Mobile phase = 4:1:1 isopropanol: acetic acid: water mixture

Define Strong acid and what kind of pKa's they will have

Strong: Dissociate almost entirely in H2O (i.e. hydrochloric acid) In the case of a strong acid, because we have almost complete dissociation into these ions, we're going to have very high concentrations of our proton and our chloride ion. This means that our Ka will be greater than 1 which means that our strong acids are going to have low pKa's.

Define chromatography

Technique used to separate molecules based on physical and chemical properties

Structure of LDH

Tetramer = 4 subunits

What are the SI units of mass and volume?

The SI unit for length is meters(m), for mass is kilograms(kg), for volume is cubic meter(m^3), for density kilogram per cubic meter(kg/m^3), for time is seconds(s), and for temperature is kelvins(K).

Why is the secondary antibody able to recognize the primary antibody?

The reason that our secondary antibody is able to come in and recognize our primary antibody is because our primary antibody, if you remember from the previous slide, was made in mice. Our secondary antibody was actually made in rabbits. The rabbit was treated with antigen from the mouse. And so our rabbit antibody is going to recognize our mouse antibody. Attached to our secondary antibody is then our detection signal, our alkaline phosphatase.

Describe the setup for the western blotting assay

The sandwich was necessary because we wanted to transfer our proteins from our gel to this membrane. The membrane has then been incubated with a blocking solution. This blocking solution contains milk. So once the membrane has been treated with the blocking solution it is then washed with TTBS and this is where we come in and start. Once we've washed the membrane we're then going to add on the primary antibody. Remember that there are going to be three different antibodies, wso we need to put the right primary antibody on the correct blot. Once we've incubated with our primary antibody, our primary antibody is going to recognize our protein of interest. So in this first step here. Then we are going to wash with TTBS three times to get rid of excess primary antibodies. We then add our secondary antibody, which recognizes the primary antibody. We are going to wash again 3 times with TTBS getting rid of excess secondary antibodies. If you remember from the previous video, our secondary antibody is also attached to alkaline phosphatase and this is what we're going to be using to visualize our proteins. So in order to have a color appear we need to add in a detection reagent. This detection reagent has substrates for alkaline phosphatase and once these substrates interact with our enzyme we're going to get a purple color appearing. However, this reaction could go on and on and we want to stop the reaction with EDTA as soon as purple bands are visible.

Western blot: Attaching the secondary antibody

The secondary antibody is going to attach to the primary antibody and so now we have our protein of interest, a primary antibody, and a secondary antibody all attached to our membrane

Describe the conversion of p-nitrophenyl phosphate to phenolate ion

This week we will be using p-nitrophenyl phosphate. What happens when we add in our enzyme is that we go from p-nitrophenyl phosphate to the p-nitrophenol and a removed phosphate group. The enzyme removes a phosphate group and works at a high pH or in an alkaline environment. Now, since this enzyme likes to work at a high pH, our reaction will continue to be at a high pH. So this p-nitrophenol will become this phenolate ion. We now have a yellow molecule and we can take it and put it back in the spectrophotometer.

Amino Acid Titration curve

Titration curve; we will generate one for our lab report Y axis is pH X axis is mL of 0.1 N NaOH Note that as NaOH volume increases on the X axis, pH increases on the Y axis. X axis is the independent variable Y axis is the dependent variable; change in pH is dependent on the mL of 0.1 N NaOH Two pKA values; transition points where we go from a protonated states to a deprotonated states. → Note that pKa 1 is fully protonated. As we transition past pK 1 which is a pH of 2.1 we move into pK 2, which is a fully deprotonated state at pH 9.9.

How to calculate pI

To calculate pl, average the two pKa values where the amino acid goes from + → zwitterion and from zwitterion → - . (In the case of glycine you average our pKaC and our pKaN. pI = (pKa1 + pKa2)/2

Elution at 280 nanometers

To start with, let's look at our first wavelength, 280 nanometers. We're looking for preotins being present, so our two proteins are ovalbumin and cytochrome c. We want to figure out in which fraction these proteins are located. So look for peaks at 280 nanometers. There are two peaks, which indicates which fraction these proteins of interest are located at.

Total volume

Total Volume (Vt): Very small protein beneath our fractionation range that is going to get stuck as much as it can in these processes and this protein will come out at the very end of our column and it will give you a value for the total volume of the column. In this experiment you will be using vitamin B-12 as the small molecule to figure out total volume.

Beer-Lambert Law

Used to relate the concentration of colored solutions to the amount of visible light they absorb. Beer lambert law: A = εbc → Allows us to understand the relationship between absorbance and concentration. → ε = extinction coefficient (M^-1cm^-1) → b: Length of light path (cm) (usually 1 cm) → C: Concentration of a substance (M)

Affinity Chromatography

Utizes specific interactions between molecules: --> receptor ligan --> antigen antibody Interactions could be varying in nature: ionic, hydrophobic, hydrogen bonding

What is vmax?

Vmax = maximum velocity of the reaction; this will be in units of molar per second^-1

Void volume

Void Volume: Based on a protein larger than fractionation range; In this experiment we are using dextran blue, which is larger than that fractionation range.

Western Blot: The process of attaching the primary antibody

We are going to be treating these membranes, which our protein of interest are now on (that was the process of this sandwich we set up; so an electrical current was applied to the sandwich that we set up and our proteins of interest actually traveled from our gel onto this membrane.). All we have now is our membrane, which has our protein of interest. The first thing we're going to do is treat our membrane with our primary antibody. This is shown here by this blue y shape. Our primary antibody is going to recognize our protein of interest so we're going to allow that to incubate together and so our primary antibody is going to find our protein of interest. Well then wash away an excess primary antibody and well treat the membrane with a secondary antibody, which is the pink shape here

What is the Fc domain?

We have the FC domain and this is the part that is going to bind to our secondary antibody. So when our secondary antibody comes in, we have the fab domain of this secondary antibody recognizing the FC domain or this bottom part of our antibody from the primary antibody.

Describe the energy barrier enzymes must overcome

We know that to get from reactants to products there is some type of energy barrier that the reaction has to overcome before it can get to products. Blue line is showing an activation energy for a certain reaction while the white line is showing that activation energy once an enzyme has been introduced. The way enzymes help reactions move forward faster is that it lowers the energy of activation.

What is attached to the secondary antibody?

We should remember where we've seen alkaline phosphatase before. So alkaline phosphatase at the very end, was going to treat our membrane with an alkaline phosphatase substrate. This substrate is going to produce a color and it is going to allow us to use this as a detection signal so we will be able so see where exactly on our blot our protein of interest is

Monoclonal vs. polyclonal antibodies

We start by using mice. Mice are given these different antigens which have different epitopes on them. The epitopes in this case are cytochrome c, ovalbumin, or Beta-galactosidase. If we were making polyclonal antibodies, once the mouse has been injected with an antigen we just isolate the serum and in the serum we'll have these poly-clonal antibodies for all the different epitopes that are of interest. If instead we isolate the spleen cells from the mouse we can then get plasma cells and when we mix these plasma cells with myeloma cells we can then hybridize them and form hybridoma cells. THe hybridoma cells are representative of the different antigens we've treated the mouse with and we can then separate these different cells and these will give us our monoclonal antibodies.

Comparison of Michaelis Menten and Lineweaver Burk

We use Michaelis - Menten because we have the technology to generate a nonlinear aggression to fit the data more accurately because when we transform it to lineweaver burk by taking the reciprocal of both sides, we have a lot of issues at low substrate concentrations. → With lineweaver burk: We have a larger Km when there's an inhibitor and then without an inhibitor while the Vmax for both are the same. So in this case, we have competitive inhibition which will contrast the lower case where the Km's are the same while the Vmax's are different. V amx is smaller in experiment with inhibitors and therefore this is what we call noncompetitive inhibition. So from a lineweaver burk we can tell where things intersect and what kind of competition they have. → When we look at a Michaelis - Menten plot, we have to find Vmax by estimating visually where vmax is and then we have to estimate where Km is. BEcause we have to estimate this visually, it takes us another step to analyze the data through nonlinear regression to determine Vmax and Km.

LDH expression

We will be loading an E.Coli lysate onto our column that has LDH. This LDH has been cloned into E coli and overexpressed. Our LDH protein has been tagged with histidine residues (which our protein of interest, LDH, is attached to) which will interact with the Nickel-NTA column. We end up with a protein of interest, attached his tag which will then interact with the column.

Define Buffers

Weak acids or bases that can react with strong acids or bases to prevent sharp, sudden changes in pH When a strong acid is added to the solution theH+ reacts with A- to create HA, shifting the equilibrium to the left When a strong base is added, OH- reacts with H+ to create water and the acid salt (conjugate base)

Define weak acid and what kind of pKa's they will have

Weak: Don't dissociate as completely in solution → We won't have as high of a concentration of our ions, so our Ka is going to be less than one, resulting in weak acids having high pKa's. See image in notes

What specifically does an enzyme do?

What an enzyme does is increase the rate at which equilibrium is attained.

Measuring in vitro antioxidant activity: Superoxide Scavenging Assay

What we are doing in the lab is measuring in vitro antioxidant activity and we will do this by using a superoxide scavenging assay. The first part of the assay we're going to be adding NADH in its reduced form and react it with O2 and a regent called PMS. PMS is going to be in its oxidized form and when this reaction goes forward we will get NAD+ in its oxidized form, PMS in its reduced form, and the generation of 2O2- and hydrogen. This 2O2- is important because this is the super oxide. What's important for the second part of the assay is the production of the superoxide. In the second part, we have our superoxide and its going to react with NBT in its oxidized state. When this reaction moves forward we get O2 and we also get NBT but in its reduced state. This NBT in its reduced state is going to be blue in color. This blue color can be monitored at 560 nanometers so we will be monitoring the formation of NBT in its reduced state with our spec.

when pH = pKa, then

When pH = pKa there are equal amounts of weak acid and conjugate base. **Greatest buffer capacity when at the pKa.**

In this lab we are testing....

When performing the antioxidant lab, you will test the ability of substances to inhibit the reduction of nitro blue tetrazolium (NBT). Fruits and fruit juices are high in antioxidants In this lab, you will determine which beverages are the best antioxidants (free-radical scavengers) by testing their ability to inhibit the reduction of nitro blue tetrazolium (NBT). In

What's the pI of Glu-Gly Lys? --> Identify ionizable functional groups

When protonated, what charge does each functional group carry? When deprotonated? → So in this case we have two carboxyl groups. These can either be COOH or COO-. So when we have a protonated group, were going to either COOH, were protonated and this will carry a neutral charge as opposed to if we deprotonate the group, which is what's shown here, well have our COO- and have an overall negative charge. When our amine groups are protonated (blue) we have NH3+ groups, giving a positive charge. However, when we deprotonate these groups, we'll have an NH2 group and this has a neutral charge.

What's the pI of Glu-Gly Lys? --> Terminal groups of the tripeptide

When we have a tripeptide, each amino acid doesn't have its own terminal group. So our peptide overall will have N terminal group and a C terminal group. But each amino acid itself will not have its own terminal group. These groups together join together to form this peptide so they're not going to be ionizable side chains. But we want to identify which ones will be. Red circle on the left is the side group of Glu, the upper left blue circle is the N terminal of the peptide, the upper right red circle is our C terminal, and the lower right blue circle is the R group on lysine. So these are the rou ionizable side chains, or functional groups, that were interred in when analyzing this tripeptide. Gly doesn't have an ionizable R group.

Initial Rate plot

X axis = time Y axis = Absorbance at 400 nm

line weaver burk plot

Y-intercept is 1/Vmax and X-intercept is 1/Km

Define buffer

a substance that can maintain the pH of a solution at a relatively constant point Solution of weak acid and conjugate base which resists changes in pH A buffer resists change in pH → Buffer is a solution made up of a weak acid and a conjugate base

Define Gel filtration chromatography

a type of column chromatography that separates proteins based on their size using size-exclusion beads; also called size-exclusion chromatography → Gel filtration; based on size

Standard curve plots?

absorbance as a function of solution concentration

Acetic acid has a pKa of 4.76. At what pH would an acetic acid buffer have the maximum buffering capacity?

at a pH of 4.76 we would have the maximum buffering capacity because we have equal amounts of our weak acid and conjugate base present.

Remember: pH > pKa

group is deprotonated

Remember: pH < pKa...

group is protonated

We are going to add a sample buffer to our samples. What does it contain?

n our sample buffer, there are a number of different components that will help our sample correctly travel down the gel. There is going to be a Tris CL buffer. This allows us to keep the pH around 6.8. The next component is SDS. SDS will linearize our protein and will give our protein an overall negative charge. The next thing in our sample buffer is beta mercaptoethanol. This is abbreviated BME. BME is going to break any disulfide bonds in our protein but the thing about BME is that it's really sticky, meaning we have to work with a sample buffer in our hood. The next thing is glycerol. Glycerol will increase the density of our sample. The reason we want to increase the density of our sample buffer is to make loading the sample into the well easier. When we load our sample into the well, it settles well. The final component is Bromophenol blue. It's blue in color and allows us to track our samples as they travel down the gel, so this lets us know when our sample is completed.

If our pH is greater than our PI

our molecule will be negatively charged.

So if our pH (of environment) is less than the pI,

our molecule will be overall positively charged.

Describe p-nitrophenol at different pH's

p-nitrophenol is colorless at a low pH = unionized p-nitrophenol is now a phenol ate ion with a yellow color at a high pH = ionized.

Henderson-Hasselbalch equation

pH = pKa + log [A-]/[HA] Therefore, you want to choose a buffer that has a pKa closest to your desired pH. NOTE:The Henderson-HasselbalchEquation is only applicable for acids with a pKagreater than 2.

Pl or isoelectric point

pH at which the net change of the amino acid is zero (neutral charge). Amino acid is entriely in Zwitterion form. If the amino acid does not have an ionizable side chain, it will be mostly in zwitterion form at neutral pH.

What's the pI of Glu-Gly Lys? --> Identify all pKa values and order from smallest to largest

pKa chart

What is pOH?

pOH: Negative log of our hydroxide (OH-) concentration

WE used a pierce 660 nm assay to...

pierce 660nm protein assay which will require us to make a standard curve using BSA and to find the concentration of our samples from GFC and IEC.

Lactate dehydrogenase (LDH)

pyruvate + NADH ↔ lactate + NAD+, present in most tissues, marker of cell damage/death Converts pyruvate to lactate - reversible, oxidation reduction reaction Conversion to lactate favored in aerobic conditions Co-enzyme NAD+ formed in forward reaction, required for glycolysis Reverse reaction occurs in the liver, NAD+ is converted to NADH

competitive inhibition

substance that resembles the normal substrate competes with the substrate for the active site

Michaelis-Menten equation/plot

v = (vmax [S])/(Km + [S]) On the X-axis we have substrate concentration and on the Y axis we have velocity of the reaction

Once you've added the sample buffer to the sample....

we heat it at 90 degrees to further denature a protein. Then we centrifuge our sample to make sure that anything on the side of the tube makes it to the bottom so we know we are adding this homogenous sample to our wells.

What is enzyme catalysis and how do enzymes carry this out?

→ A catalyst is a substance that increases the rate of a reaction → Enzymes do this by reducing the energy of activation of a particular reaction, and there by accelerating the formation of products from reactants and vice-versa.

What is absorbance? Does it have units?

→ Absorbance (A): -log(T) Does absorbance have units? No it doesn't because all units of items involved get crossed out.

Describe the extinction coefficient and what it is dependent upon/what is dependent on it

→ Absorbance depends on ε → ε is unique to each substance → ε depends on the intrinsic properties of the substance being measured → Example: Peptides; ε is largely dependent on aromatic residues such as tryptophan and tyrosine.

Define affinity chromatography

→ Affinity Chromatography; Based on specific interactions between molecules

How does gel electrophoresis work in terms of electricity?

→ An electric field is also applied to the gel → In which direction (towards the + or - electrode) do the proteins run and why? → The electric field will apply a negative charge at the top of the gel and add a positive charge down at the bottom of the gel.

Why is BME important?

→ BME breaks the disulfide bonds, removing tertiary and quaternary structure of our protein sample. → If the cysteine residues form disulfide bonds, we're either going to get some sort of tertiary structure where our protein is coming together in a different way or we get some quaternary structure where we're having other protein chains come together. SO BME is going to break any of these disulfide bonds that form. This is important because it could allow us to create a more linear protein. SO if we have these disulfide bonds in our protein, we want to make sure that it's completely linear when running it on an SDS-PAGE gel. → In both lanes, we have the same protein sample, however, one lane has been treated with BME. So the second lane that we've added BME is because in this first lane, we have two different protein species, a larger species and a smaller species. This is indicating we have multiple proteins coming together to increase this sample's molecular weight. When we add the BME, our disulfide bonds are getting broken and separating the protein chains, creating a single species and a smaller molecular weight.

Will glutamic acid be positively charged or be negatively charged at pH 6? What about arginine?

→ Glutamic acid has a pI of 3.22. Since pH > pI, our molecule is going to be overall negatively charged. → Arginine acid has a pI of about 10.76. Since pH < pI, our molecules are going to be overall positively charged.

Goal of the separation of protein part 1

→ Goal: Separate a mixture of ovalbumin and cytochrome C using ion exchange chromatography (IEC) and Gel filtration chromatography (GFC).

Describe Ion exchange chromatography stationary phase

→ IEC retains molecules based on ionic interactions (Ionic interactions are simply charge-charge interaction) → Stationary phase contains ionic functional groups which attracts molecules of opposite charge → Further divided into... - Cation exchange chromatography - Anion exchange chromatography

Changing the salt concentration of the buffer

→ In this lab we will have a resin that is negatively charged and our protein will be positively charged. Because the proteins are positively charged, there will be an interaction between the protein in the resin. However, this week, to elute off that protein, we'll be changing the salt concentration of the buffer. So when we increase the salt concentration the column is going to be flooded with positively charged sodium ions. When the column gets flooded with these sodium ions they are going to compete off the protein, and the protein will elute from our column. These sodium ions will displace the protein and the protein will be eluted out. Because were using the same buffer just with different salt concentrations, its important to take the right buffer at the right time. → We will use salt concentration to elute out the molecules instead of changing pH → The Na+ ion will displace the protein and cause it to elute out of the column → Cytochrome c pI = 9.5 → Ovalbumin pI = 6

GFC matrix selection

→ Matrices (pores/beads) have different fractional ranges, or the range of molecular weights the column can accommodate. → Should include the molecular weight of protein of interest → If the protein of interest is 60kDa, which column would you use to isolate it? The second one. Because the first column 60 is right on the edge of that fractionation range. → You want to make sure when picking a column that your protein of interest is right in the middle of that range.

Define Cation exchange chromatography

→ Negatively charged stationary phase → Binds positively charged molecules (cations) → Repels, and elutes, negatively charged molecules → Removal of bound ions (negatively charged) by changing salt concentration or pH

Separation of Protein Lab - 3 parts

→ Part 1: Separate mixture of proteins based on chemical and physical properties (molecular weight, Kw) using gel filtration and ion exchange chromatography. Purifying two proteins either using gel filtration of IEC. → Part 2: Using separated proteins from week 1 you will quantify (determine the concentration and define chemical (extinction coefficient) and physical properties (size)) using SDS-PAGE. Determine concentration of protein. → Part 3: Using separated proteins from week 1 determine the concentration of proteins using a Pierce 600 nm Protein Assay and confirming protein identity through a western blot.

Define Anion exchange chromatography

→ Positively charged stationary phase → Binds negatively charged molecules (anions) → Repels, and elutes, positively charged molecules → Removal of bound ions by changing salt concentration or pH

Why is SDS important for protein preparation?

→ SDS coats protein backbone with a negative charge and linearizes the protein (due to long tail), removing secondary structure → This makes sure that separation of denatured protein is only based on size. → If we have a folded peptide in secondary structure. If we take this and add SDS onto here, our protein is going to linearize and this is because SDS coats the backbone of the protein and it's going to much more linearize our protein and cause it to have an overall negative charge.

What does a change in pH of our buffers allow us to do?

→ The functional group of each amino acid can vary in charge, dependent on the surrounding environment → A change in pH can alter the charge of an amino acid's functional group → By increasing pH of our buffers, you can selectively elute each amino acid via IEC; If we have a positive charged stationary phase, our negatively charged amino acids are going to stick, where our positive ones are going to flow out. So by increasing the pH and changing the charge of the amino acids, these amino acids will either stick or come out.

How does gel electrophoresis work?

→ The gel is comprised of a gradient of Polyacrylamide, which helps to separate molecules by their size. → At the top, we have our protein wells, which is where we will load our sample. Up at the top where our wells are, we have our stacking gel. Our stacking gel is a little bit more rigid than the rest of our gel and allows us to easily input these samples in our wells. → At the bottom, the rest of our gel is known as our resolving gel. The resolving gel actually has a gradient of polyacrylamide. It does from about 4% to about 20% near the bottom; concentration increases. → When we load proteins of different sizes, different proteins are going to be able to go through the pores that our polyacrylamide creates. → So our larger proteins won't be able to travel through those pores as much and they'll end up toward the top of our gel. → As we smaller proteins, these proteins will be able to travel through the polyacrylamide more easily and make it further down the gel. So, our larger proteins are trapped by the pores and they travel less. Our smaller proteins fit through the pores and travel further.

Gel filtration Chromatography in detail

→ The stationary phase is made up of beads with pores of different sizes. So different proteins depending on the size will either get trapped in those pores or they won't get trapped in those pores. → Small proteins are going to get caught up in all of the pores in the ebads. The yellow circles are larger proteins. So larger proteins are not going to get stuck up in those beads and they are going to elute much more quickly than the smaller proteins.

What is the charge of this peptide at pH = 1?

→ Think about the different R groups on each amino acid. Depending on whether the amino acid is protonated or deprotonated will result in a different charge on each of these R groups. → Arginine, because it has either an NH3+ group on it or a NH2 group on it, it can either be Positively charged or neutral depending on the pH you're at. → Lysine can be positively charged or neutrally charged depending on the pH → Cystine has this SH group on it. Depending on pH, cystine can be neutrally charged or negatively charged. → Glutamate can either be neutrally charged or negatively charged because of this COOH group or COO negative group. → Histidine can either be positively charged or negatively charged because of that NH2 group or NH3+ group on it. The next thing you want to do is figure out your pKas for all of the different titratable groups in your peptide. Although we have all of the R groups of our amino acids here in our peptide, we also want to remember that on a peptide we also have an amine N-terminal and a carboxyl C-terminal. So we also have to take these pKa's into account. Remember: pH < pKa - group is protonated , pH > pKa - group is deprotonated View images on notes

E.Coli Lysate to demonstrate western blot advantage:

→ Today you will run lysate from E.coli that over-express 3-galactosidase; note, an E.coli lysate is the contents of an E.coli cell. → These E.coli make an excess amount of the enzyme that breaks down the sugar B-galactose. (this e coli churns out a ton of beta galactosidase so there's a high level of protein in these cells.) → E.coli are lysed to spill the contents of the cells → When ran on a gel, you will see the total proteins expressed by the cell at the time of lysis → Over expressed proteins will appear darker than other bands on the gel (more protein, so darker; it's hard to tell which bands are which) → Wetsern blotting allows for you to determine if your protein of interest is in your lysate, when it's difficult to determine from the gel through treatment with a beta-galactosidase antibody. → Allows us to identify where our ovalbumin is, where our cytochrome c is, and where the beta galactosidase band is on our gel.

Describe the different kinetic parameters

→ Vmax (maximum velocity): it represents the maximum rate attainable → Km (Michaelis - Menten constant): Km is a measure of the stability of the ES complex (the larger the Km, the less stable the ES complex); Km = Vmax/2 → Kcat (catalytic constant/turnover number): The turnover number of an enzyme is the number of substrate molecules for moles of substrate that are converted to product per unit time, when the enzyme is fully saturated with the substrate; Kcat = Vmax/[Et] in units time^-1. This is the maximum rate or the maximum level of product formation that can occur per mole of enzyme per unit time. → Kcat/Km (catalytic efficiency): A large ratio favors the formation of products and vice versa. If we have a small ratio, the reaction favors the reactants. This is in units of molar^-1 times sec^-1

IEC review

→ We separate proteins based on charge. So the stationary phase can either be negatively charged or positively charged. And it will attract proteins of the opposite charge while proteins of the same charge will pass right through the column because they will be repelled by the stationary phase. → How did we elute each molecule? Utilized buffers to change pH to manipulate the charge on different amino acids and allow them to elute out of the columns at different points.

Amounts in solution (%w/v, %v/v )

→ mg/ml → %w/v = amt of substance dissolved in 100mL final volume → %v/v = amt of a liquid mixed with water for a 100mL final volume

Review of the isoelectric point

→ pI or isometric point: pH at which the net charge of the amino acid is zero (neutral charge) → When the net charge of our amino acid is zero, or has a neutral charge, this by definition is the zwitterion form. → If the amino acid does not have an ionizable side chain, at neutral pH its zwitterion form will predominate. → At a low pH, we have a lot of H+ floating around in the environment. So since there is a lot of H+ in the environment, at low pH our amino acid here is going to hang on to its H+. → At a high pH, we have a lot of OH- around in the environment. So now, our glycine will want to donate the H+'s that it has. So, the charge of glycine here is going to be overall negative.