BioChem Lab Midterm

How would you make a carbonate buffer if your stocks were 0.5 M instead?

(0.5)(x)=(0.1M)(100ml) x=20 ml

if 2.0 mL of 10 M NaOH is added to a liter of water, what would the final pH be? Give your answer to two decimal places.

(10 M)(0.002mL)=(x)(1.002L) x=0.01996 M pOH=-log(0.01996 M)=1.7 14-1.7=12.3

your second unknown protein has an absorbance of 0.8. how much protein is in this sample?

0.8=0.06x x=0.013 msg/mcl

Match the items. a. breaks disulfide bonds b. breaks hydrogen bonds and electrostatic interactions c. Gives a uniform negative charge and disrupts hydrophobic interactions 1. Mercaptoethanol 2. SDS 3. pH and heat

1. a 2. c 3. b

why do we add SDS to the sample?

1. breaks hydrophobic interactions 2. creates overall negative charge

if a solution of BSA contains 2mg/ml, 5 ml contains what mass of BSA?

2mg/1ml=x/0.005ml= 0.01mg

which protein would travel farther? 120kDA or 55kDa?

55kDa

what are the two chemicals that cause the polymerization reaction

APS TeMED

the assay used in the lab was

BCA

What does the slope of the line for each concentration of substrate represent?

Vo

You perform your experiment as described above. Your first set of data (for substrate concentration = 0.5 mM pNPP) is fit by a trend line with the equation y = 0.032x + 0.017 What is the Vo for this concentration of pNPP? a. 0.032 b. 0.0642 c. 0.017 d. 0.5/0.032

a. 0.032

To elute target proteins from a hydrophobic chromatography column, which of the following conditions would be the most appropriate? a. Low salt concentrations b. High salt concentrations c. Adding a soluble ligand which competes with the affinity tagged protein for binding to the column d. Just keep washing buffer through the column, isocratic elution

a. Low salt concentrations

Use the Henderson Hasselbalch equation to calculate the volume in mls of a) acetic acid and b) acetate (the congugate base) to use to make 100 ml of a 0.01M acetate buffer, pH 5.0, from 0.01M stock solutions. pKa acetate = 4.76. a. acetate = 63.5 ml acetic acid = 36.5 ml b. acetate = 50 ml acetic acid = 50 ml c. acetate = 36.5 ml acetic acid = 63.5 ml d. acetate = 68.4 ml acetic acid = 31.6 ml

a. acetate = 63.5 ml acetic acid = 36.5 ml

What color change occurs when proteins combine with Coomassie dye under acidic conditions? a. brown to blue b. blue to brown c. yellow to red d. blue to purple

a. brown to blue

Your peptide has the primary sequence Glu-Gly-Asp-Glu-Asp-Phe- Ala. What type of colum would you chose to purify it in a buffered solution at pH 8? a. positively charge anion exchange column b. negatively charged cation exchange column c. hydrophobic column

a. positively charge anion exchange column

Using BSA concentrations of 0 mcg/mL, 1 mcg/mL, 3 mcg/mL, 4 mcg/mL, and 5 mcg/mL you get the following absorptions: 0.02, 0.12, 0.32, 0.42, 0.52. (mcg = microgram) What is the standard curve equation for this BSA standard? a. y=0.1x+0.02 b. y=10x-0.02 c. y=0.02x+0.1 d. y=0.02x-10

a. y=0.1x+0.02

If there is only a very small amount of light being detected, the spectrophotometer reading is not accurate and cannot be used. Very little light is detected when there is a ............... absorbance. a. high b. low

a.high

what is the protein we detected? what is the reaction that we will perform to detect the protein?

amylase; addition of starch for amylase to break down to maltose

As in the previous question: Using BSA concentrations of 0 mcg/mL, 1 mcg/mL, 3 mcg/mL, 4 mcg/mL, and 5 mcg/mL you get the following absorptions: 00.2, 0.12, 0.32, 0.42, 0.52. You dilute your sample 1:5 and get an absorbance of 0.15. What is the concentration of your sample? a. 1.3 mcg/ml b. 6.5 mcg/ml c. 0.035 mcg/ml d. 0.175 mcg/ml

b. 6.5 mcg/ml

You are planning an experiment that you want to keep buffered at pH 5.0. Which buffer(s) from the list above could you use? a. Glycine and phosphate b. acetate and citrate c. phosphate and carbonate d. any of the buffers in the table will work e. Tris or phosphate buffers f. All of the buffers named above

b. acetate and citrate

At pH 7.4, an acetate buffer is predominantly in what form? a. acidic (HA) b. basic (A-) c. equally acidic and basic

b. basic (A-)

Maltose is the product of the amylase reaction. When maltose reacts with the dye we add to the amylase reaction, it turns dark yellow. The reaction containing the fraction corresponding to the highest amount of amylase will be: a. pale yellow b. dark yellow c. the first fraction eluted from the column d. the last fraction eluted from the column

b. dark yellow

What is the path of light through a spectrophotometer? a. photodetector, filter, sample, light source b. light source, filter, sample, photodetector c. light source, sample, filter, photodetector d. filter, sample, photodetector, light source

b. light source, filter, sample, photodetector

Larger proteins run .............. than small proteins a. faster b. slower c. further

b. slower

After we elute the proteins from the column, we will measure the activity of amylase to detect which fraction contains the enzyme. What is the substrate for the reaction? a. amylase b. starch c. cellulose d. maltose

b. starch

Protein separation techniques are often based on the following properties EXCEPT: a. solubility of the protein b. viscosity of the protein c. charge of the protein d. specifi binding affinity of the protein

b. viscosity of the protein

You determine that your solution is too concentrated to measure the absorbance. You reach this conclusion because your absorbance was: a. 0.05 b. 1 c. 2.3

c. 2.3

A 1mg/ml solution of BSA contains ........ mg of bovine serum albumin in 20 microliters a. 20 milligrams b. 10 milligrams c. 20 micrograms d. 0.02 micrograms

c. 20 micrograms

Suppose an acid HA has a dissociation constant Ka=1×10−1. It is mixed into a buffered solution, and its equilibrium concentration [HA] = 0.1M. If the concentration of its conjugate base is 10M, what is the pH of the solution? a. 1 b. 2 c. 3 d. 8

c. 3

How many points will each of your time course graphs have (including the time zero time point)? a. 4 b. 5 c. 6

c. 6

Glacial acetic acid (pure acetic acid) has a concentration of 17.54 M. If 85.5ml of glacial acetic acid is diluted to 250ml, what is the acetic acid concentration in the final solution? a. 0.17M b. 1.75M c. 6.0M d. 7.5M

c. 6.0M

We will use which assay in our lab? a. Bradford b. General Protein c. BCA

c. BCA

How will you determine the Vo (the y-axis value) for your Vo vs [S] graph? a. By measuring the Vmax of the reaction b. By calculating the concentration of pNitrophenol in each reaction c. By getting the slope of the [P] vs time plots d. From the absorbance values for the experiment

c. By getting the slope of the [P] vs time plots

How is a protein eluted from an ion exchange? a. By increasing the pH of the mobile phase b. By decreasing the pH of the mobile phase c. By increasing the salt concentrion of the mobile phase d. By decreasing the concentration of the mobile phase

c. By increasing the salt concentrion of the mobile phase

The purpose of a standard protein is to: a. compare the unknown to one concentration of the standard b. construct two curves: a standard and an unknown c. construct a standard curve to use to compare the unknown to d. see whether the assay works or not

c. construct a standard curve to use to compare the unknown to

Which of the following buffers would be appropriate to use in this experiment? a. acetate b. sulfonate c. phosphate d. any buffer will work

c. phosphate

How will we measure the rate of the reaction? a. substrate consumption over time b. substrate consumption and product formation over time c. product formation over time d. enzyme consumption over time

c. product formation over time

how will we elute our protein today?

change salt concentration

If less than 1% of the light entering the sample passes through to the detector, the reading is not reliable. Using the formula Absorbance = log (light entering sample/light leaving sample), calculate the absorbance when 1% of the light passes through the sample. a. 10 b. 0.1 c. 1 d. 2

d. 2

You have a tissue extract containing two proteins. One has a molecular weight of 60,000 daltons. The other has a molecular weight of 20,000 daltons. You put your extract onto a size exclusion chromatography column. Which peak in your results below corresponds to the 20,000 dalton protein, and how do you know? a. Peak 1 because the bigger protein elutes first b. Peak 2 because the bigger protein elutes last c. Peak 1 because that peak is the biggest. d. Peak 2 because the smaller protein elutes last

d. Peak 2 because the smaller protein elutes last

Calculate the Km and Vmax for the experiment shown in the graph below. a. Vmax = 2 units/ml/min Km = 0.02 mg/ml b. Vmax = 27.46 units/ml/min Km = 0.5 mg/ml c. Vmax = 0.0002 units/ml/min Km = 0.0004 mg/ml d. Vmax = 5000 units/ml/min Km = 2 mg/ml

d. Vmax = 5000 units/ml/min Km = 2 mg/ml

Protein purification refers to: a. separation of proteins from other biomolecules b. separation of a particular protein from contaminating proteins c. purification of proteins d. all of the above

d. all of the above

The absorbance of a solution can be used to calculate: a. color b. density c. type of solute d. concentration

d. concentration

The reason for adding SDS to the sample is to a. denature the sample (together with mercaptoethanol and heat) b. promote protein folding and therefore function c. give the sample a positive charge d. give the sample a negative charge e. a and c f. a and d

f. a and d

What type of chromatography did we use?

hydrophobic column

in the lab's column, what property allows our protein of interest to attach to the column

it is hydrophobic

which component sample reduces disulfide bonds?

mercapethanol

Aspirin has a pKa of 3.4. What is the ratio of A¯ to HA in: (a) the blood (pH = 7.4) (b) the stomach (pH = 1.4)

pH = pKa + log [base]/[acid]

what will be the pH of the carbonate buffer you made above be if you then add 0.005 moles of NaOH(strong base). Assume the addition the sodium hydroxide has no effect on the volume.

pH=10.3+log[0.1M+0.05M]/[0.1M-0.05M] pH=10.78

which gel compresses the proteins so that they enter the lower gel together?

stacking

what does y represent? what does x represent?

y=absorbance [wavelength] x=concentration [mcg/mcl]