BISC 330 Lab Practical

What is the role of B galactosidase?

enzyme that catalyzes hydrolysis of disaccharide lactose, producing glucose and galactose in the process

what is relative mobillity (Rf)? and how is it calculated?

ratio of distances each protein migrates to that of a small ionic tracking dye calculated by the distance of protein migration over the distance of tracking dye migration

What is Beer Lambert Law

relationship between absorption and concentration

what does the lower stacking gel allow?

proteins to migrate rapidly and be compressed at edge of the denser resolving gel

What does michaelis menten enzyme kinetics do?

provides insight about enzyme conversion of substrate to product

What was the goal of these bullshit labs over the course of this semester?

purify B galactosidase from bacterial culture; specifically Escherichia coli strain ML-308

T/F: rate of production of oNP from oNPG is directly proportional to amount of B gal present

True

What is the goal of the calvin cycle?

fix carbon and generate glucose does not need light

You create a standard curve from known concentrations of bovine serum albumin (0-10mg) (a common protein). When you plot this on excel the resulting formula for the slope is y = 2.5x + 0.38. You measure your protein sample of interest and the absorbance is 0.7, what is the concentration of your protein?

0.13mg

You run a solution in a cuvette in a spectrophotometer and the transmittance is 65%. What is the absorbance?

0.187

If you dilute 15mL of a 1M stock solution with 35mL of water, what will be the final concentration of the solution?

0.3M

What is the final concentration if 15mL of a 1M stock solution of X is diluted with 35mL of water?

0.3M

Using the principles of spectrophotometry, given a transmittance T = 24%, what is the absorbance?

0.62

Using 0.5M of Y, what volume of Y do we need to make a 1:2 dilution with a final volume of 50mL?

25mL

You dilute XmL of a 1M stock solution to a final volume of 40mL and a final concentration of 0.16M. How much water was used to dilute the stock solution?

33.6 M

How many rounds of a 1:2 serial dilution do we perform to obtain a 0.5M solution starting from 16M of stock?

5 rounds

What volumes correspond to a reading of 0;9;8 on a P10 and a P100 pipette respectively?

9.8 ul and 98 uL

T/F: in order to preserve stability and activity of enzyme during the separation process, it is ok to be lenient with pH, salt concentration, and enzyme environment

False, enzymes are very particular to certain conditions and can denature in undesirable environments

T/F: the light reaction require sunlight and is a slower process than the Calvin Cycle

False, it is a faster than Calvin Cycle

what are the two types of ion exchanges?

cation exchanger anion exchanger

Salting In procedure

charged protein in solution with low salt concentration added. The protein is surrounded by proteins of opposite charge. The increase in solute to solvent ratioe creates a hydration shell making the protein soluble in solution

You are asked to prepare a 4 point 1:4 serial dilution from stock that is 1M

- 4 points means four dilutions - Final volume is 10 mL - ex: first solution divide by 4, next divide by 4^2, third divide by 4^3, etc; do this 4 times for four point - volume at each stage stays same, in this case 10 mL - use C1V1 equation to solve for variable

hydrophobic interaction columns

- use salting in/salting out concepts - decresing salt concentrations increases solvation of hydrophobic molecules - inverse of ion exchange

determine the 2M stock solution of X required to make 100 mL of 0.5 M solution

C1 = 2M V1 = TBD C2 = 0.5 M v2 = 100 mL

What dye is used in Bradford Assay and what is its charge?

Coomassie Brilliant Blue, anionic

two types of lipoproteins that carry cholesterol?

LDL and HDL

In gel electrophoresis the fragment at the lower end (farther from the loading well) are:

Smaller and move faster

Which of the following is not true about pipetting?

You can reuse tips on different reagents as long as they dont react

Which sample will have a higher absorbance value?

a highly turbid solution

What is SDS?

a negatively charged detergent that binds to polypeptide chains

Sitting drop method?

a part of protein crystallization where the protein solution is on an elevated platform

Serial Dilution is used when

a range of concentrations are desired when solute or solvent volumes are low to use in standard dilution intermediate steps are identical to standard dilution

gel filtration chromatography

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

What is vapor diffusion?

a way of crystallizing proteins, take a solution of sample at higher protein concentration and cause it to precipitate out of solution (similar to salting out)

What is the mechanism that allows salting out process to work?

ammonium sulfate increases high salt concentrations allowing proteins to aggregate bc their surface charges neutralize

What two gels is polyacrylamide gel made of?

and upper stacking gel and lower stacking gel

what was the charge of the ion exchange chromatography used in lab?

anion

what is an ion exchanger?

composed of gel matrix (beads) to bind proteins and a mobile phase (buffer) for protein to remain in solution

noncompetitive inhibitor

does not bind at substrate site (allosteric site) and binds to both enzyme and substrate complex

uncompetitive inhibition

does not bind at substrate site and only to enzyme substrate complex

What are the types of chromatography columns?

gel filtration chromatography and affinity chromatography

what are lipoproteins?

globular particles consisting of nonpolar lipid cores surrounded by amphipathic coat or protein, phospholipid, and cholesterol

mixed inhibition

graph appears like a noncompetitive graph but with a high affinity for binding enzyme without the substrate or enzyme-substrate complex

competitive inhibition

inhibitor binds at substrate site preventing substrate from binding

what does salting out procedure do?

involves a high increase in ionic strength leading to reduced solubility of a protein

What does O-NP do?

it is hydrolyzed by Beta gal and turns yellow in solution, it allows for detection of B gal

When are pipette pumps used?

largeer volumes (mL)

What two reactions make up photosynthesis?

light and dark reactions (calvin cycle)

what is electrophoresis?

movement of charged particles in electric field

what kind of beads will an anion exchange bind?

negatively charged proteins

What two assays were used in spectrophotometry and what was the purpose for each?

o-NP for activity of protein and Bradford Assay for quantity of substance in unknown solution

What does it mean to purify an enzyme?

perform a procedure that uses extraneous compounds such as cell debris, other enzymes, and unwanted organic molecules to separate from wanted enzyme

what is ultrafiltration?

process that uses semipermeable membranes to separate particles based on size

Main difference between salting in and salting out procedure

salting in is a mild increase of ionic strength leading to increase in solubility of a protein for it to become hydrophilic. salting out is a high increase in ionic strength leading to reduced solubility of a protein, making it hydrophobic

What was the technique used in protein extraction of B galactosidase?

salting out using ammonium sulfate

affinity chromatography

separates based on molecular affinity of protein binding to substrate

What does SDS page do?

separates protein on molecular weight NOT charge

What does column chromatography do?

separates proteins on size, charge, molecular affinity, or hydrophobic interactions

what is Cholesterol?

steroid that is a precursor to most other steroids, usually associated with lipoproteins in the blood

Where does the Calvin cycle take place?

stroma

What is kinetics?

study of reaction rates by monitoring concentration of reactant over product

The figure below shows a DNA fingerprint exhibiting samples from a murder victim, two suspects, and a sample collected from crime scene. Who is more likely to have committed the murder: Suspect 1 or Suspect 2?

suspect 1 (just match up SDS ladder)

Lineweaver-Burk Plot

takes the reciprocal of mm equations and is useful to determine km and 1/2 vmax

spectrophotometry

the process that determines the amount of light absorbed by colored compounds. it is used to estimate compounds based on color intensities

where do light reactions take place?

thylakoid membrane

Why do we measure a blank in spectrophotometry?

to eliminate absorbance readings for a sample with dye and without protein

What is the purpose to crystallizing proteins?

to study enzymes reactions and reaction sites

what do the light reactions generate?

transmembrane proton gradient for ATP formation and reducing power for NADPH production

what does HDL do?

transports endogenous cholesterol from tissues to the liver

What does LDL do?

transports endogenous triacylclylcerols and cholesterol from the liver to the tissues

When are standard dilutions performed?

when only small number of diluted concentrations are desired or if there is surplus of solute or solvent

What is the pI of a protein?

when protein has zero net charge, isoelectric point

What color is oNP?

yellow