Chem567 Aldolase Exam

the homogenization buffer is at what pH?

7.3

the small amount of B-Mercaptoethanol in the homogenization buffer is for what?

to keep proteins in a reducing environment

-this is because when the cells are broken open, proteins are exposed to an oxidizing environment, which can change the number of disulfide bonds. we want to prevent that

the small amount of EDTA in the homogenization buffer is for what?

to chelate divalent cations and reduce protease activity

why do you avoid foam during homogenization?

foam is denatured protein

in the first differential centrifugation, what is in the pellet and what is in the supernatant?

Pellet -> whole cells unaffected by buffer and connective tissue

Supernatant -> contents of whole cells that were broken open in blender

When would you add DNase?

If the solution is overly viscous, DNase can break down genomic DNA that is present and thin out the solution

-we do not add DNase in this case

What happens in Ammonium Sulfate fractionation?

proteins are kept in solution by interactions between hydrophilic portions (which are on the outside of protein) of the solvent

What are we doing when we add ammonium sulfate?

1) neutralize surface charges

2) reduce chemical activity of protein (denaturing at as concentrations increase)

3) diminishing the effective concentration of water (causing protein to precipitate out of solution)

At 40% ammonium sulfate concentration, what has precipitated?

varying proteins have precipitated, but aldolase remains in the solution with other proteins

At 60% ammonium sulfate concentration, what has precipitated?

Aldolase, along with some other proteins, have precipitated

Why do we add ammonium sulfate slowly?

to avoid locally high concentration

-take about 5 min to add it all to solution

When we centrifuge, what is in the pellet?

Pellet -> precipitated proteins, including aldolase

What do we use dialysis for in this experiment?

1) to remove salt and make the protein soluble again

2) buffer exchange

What is MWCO?

Molecular Weight Cut Off (MWCO) is the size of the holes in dialysis tubing responsible for which particles leave the concentrated solution into the buffer

Why do we need to use dialysis for buffer exchange?

We need to eliminate the phosphate buffer for the next step of the experiment

What do we use the Phosphocellulose column as?

1) ion exchange column

2) binding affinity column

What happens for the ion exchange column aspect?

our protein binds to the phosphocellulose column based on the charged attraction of the positive protein and negative phosphates

What happens for the affinity column aspect?

phosphates from the phosphocellulose column fit in the active site of our protein because fructose-1,6-bisphosphate is the natural substrate for aldolase

At what pH are we binding our protein to the phosphocellulose column?

ph = 7.3

Why do we wash the protein-bound phosphocellulose column with a buffer of pH = 7.8?

to wash off any proteins that will come off based on the charged attraction, basically eliminating the ion exchange aspect of the column

What happens when we add Fructose-1,6-Bisphosphate to the column after this?

eluting the column with FBP will un-bind our aldolase from the column since it is the natural substrate

Why do we use the Gel Exclusion Column

1) Gel filtration chromatography

2) determine native MW of aldolase

True or False: The gel exclusion column does not chemically interact with proteins at all

TRUE

If we have two peaks in our gel exclusion absorbance results, how can we differentiate which protein is which?

larger molecules elute first, smaller molecules have longer path lengths

How do we account for varying column sizes?

find the void volume (Vo) and divide our elution volume by it (Ve/Vo) for consistent results

What is the Bradford Assay for?

gives us the concentrations of protein present, or amount of protein present

When is our S1 sample from?

supernatant from differential centrifugation step

When is our S2 sample from?

after ammonium sulfate fractionation and dialysis

When is our S3 sample from?

after purifying our protein on the phosphocellulose column

When is our S4 sample from?

after the gel exclusion column

What do you plot for the Bradford Assay portion to solve for amount of aldolase?

known ug of BSA mixed with Bradford mix by absorbance, then same with our samples and the mix with absorbances measured

we find concentrations in ug/uL for each

for every 1 cleavage of aldolase on FBP, how many NADH do we need turned into NAD?

1 aldolase + 1 FBP = 2 DHAP

2 DHAP + 2 NADH = 2 NAD

Note: triosephosphate isomerase turns G3P -> DHAP

alpha-glycerophopshate dehydrogenase turns DHAP -> G3P in the presence of NADH

What is an SDS-PAGE gel?

-Sodium Dodecyl Sulfate (SDS) has a charged end and a nonpolar end

-gives a net negative charge to whatever runs through it

-denatures protein and gives a constant mass:charge ratio

-shorter proteins go through faster

Does the SDS gel break disulfide bonds?

NO it does NOT

-this is why we add large amounts of B-mercaptoethanol, to breakdown disulfide bonds and allow proteins to linearize

What does it mean if multiple bands appear on a gel lane?

either multiple proteins are present and the protein wasn't purified, or the purified protein has multiple subunits of different sizes

-aldolase has 1 band, all subunits are same size

How do we calculate Rf for each protein?

Rf = (distance migrated from where it enters the resolving gel) / (distance migrated by dye front)