BCH 454 Quiz 2

Steps prior to purifying a protein

1) Identify the source of protein

2) Choose buffer with the proper pKa for the protein's optimal pH

3) Break the cells

What are some things to look for when identifying the protein source?

The quantity needed, how abundant the protein is in the organism, how cheap it is

The range of the buffer is...

+1/-1 pH units from the pKa

What is important to consider when choosing a buffer?

The pKa and how the pKa varies with temperature; important to choose pKa that is the right range for the optimal pH at the protein's optimal temperature. Same goes to ionic strength (or how many ions are in the solution)

What is the purpose of buffer?

To make sure the solution stays stable through the purification and extraction processes

Cell disruption techniques, what they're used with, and how they work

1) Cell lysis; erythrocytes; disrupts the cells through osmosis stress

2) Enzyme digestion; lysozyme treatment of bacteria; digests the cell wall and leads to osmotic disruption

3) Chemical solubilization/autolysis; toluene extract of yeast; cell wall or membrane partially digested chemically and lytic enzymes complete the process

4) Hand homogenizer (blender); liver tissue; cells forced through narrow gap which rips off cell membrane

5) Mincing; muscle tissue; cells disrupted during mincing process by shear force

6) Blade homogenizer; muscle tissue, animal tissue, and plant tissue; chopping actions breaks up larger cells and shears smaller ones

7) Grinding with abrasive agent; plant tissues and bacteria; microroughness rips cell wall

8) French press; bacteria, plant cells; cells forced through small orifice at high pressure and the shear disrupts cells

9) Ultrasonication; cell suspensions; high pressure sound waves cause disruption of cells

10) Bead mill; cell suspensions; rapid vibrations by glass beads rips cell walls off

11) Manton-Gaulin homogenizer; cell suspensions; cells forced through small orifice at high pressure and the shear disrupts cells (like French Press but on a larger scale)

Which cell disintegration processes are the most gentle?

Cell lysis, enzyme digestion, chemical solubilization/autolysis, hand homogenizer, mincing

Which cell disintegration processes are moderate in intensity?

Blade homogenizer, grinding with abrasive

Which cell disintegration method processes are the most vigorous?

French press, ultasonication, bead mill, Manton-Gaulin homogenizer

Which cell disintegration process is used for this lab's protein purification experiment?

Ultasonication

What are steps to get the cells to a usable state after a mixture of broken cells and buffer (name?) is obtained?

Centrifuge the crude homogenate to obtain a supernatant and pellet (soluble proteins will be found in supernatant and membrane bound will be in pellet). If in supernatant, process is done until concentration. If in pellet, resuspend and use either: sonication, alkali, detergents, phospholipase, or acetone

How are the methods done to membrane-bound proteins in pellets all different?

Sonication can destroy cells so it must be done carefully; alkali agents and detergents dissolve the membrane but has to be non-ionic for it to work; phospholipase dissolves membrane enzymatically; and acetone dissolves it then evaporates

What process is used on a crude homogenate if a desired protein is in an organelle?

Purify organelle through differential centrifugation (centrifuging at 1000XG and then centrifuging the supernatant again at 18800XG to get a pellet of organelles). The organelles can be subjected to sonication, alkali or detergents, phospholipase, or acetone

Density gradient centrifugation

Can be used to seperate the crude homogenate based on density. Start with a solution containing 5% sucrose and 30% sucrose, centrifuge at 100,000XG, and the proteins would band according to density. This can then be fractionated

What assays must be done prior to purification? Why?

A total protein assay (such as BCA, Lowry assay, Bradford or A280) allows to detect total protein concentration. Specific activity assay (such as Enzyme assay) allows to determine units of enzyme activity per total protein; the purer the protein, the higher the amount of enzymatic units

Which total protein assay is most reliable? Why? Which is used in this lab?

BCA assay because it does not vary from protein to protein; A280 is used in this lab

What must be done right before purification?

Concentrating of protein

What is the purpose of the concentration step?

It allows to have the protein as concentrated as possible before the actual purification step so it is easy to purify and has as little contamination as possible

Methods of concentration

1) Ammonium sulfate: a typical first step in purification and can also be used as a concetrator; most common

2) Lyophilization: freeze drying to remove anything other than the protein

3) Ultrafiltration: membrane filter with a specific probe for the molecular weight needed (mw of the protein)

4) acetone: precipitates into a powder that can then be rehydrated during purification

5) Dialysis: high concentration of solute so it draws liquid out of bag through osmosis

What is salting out?

Also known as ammonium sulfate precipitation if that is the salt used, it is process typically used as the first step of purification. It works by hydrophobicity, with increasing salt concentrations making the hydrophobic interactions between water and the proteins stronger. Once the hydrophobic interactions are strong enough, the protein precipitates. Each protein percipitates differently

What does salting out achieve?

Partial purification

What are the axes on a salting out curve? What is it used for?

% solubility on the y axis and % saturation of ammonium sulfate on the x axis; the % saturation can be used in chunks as "cuts". This is a way for scientists to take out everything that precipitates in a certain percentage bracket and leave out everything outside of there that does not precipitate

What is ultrafiltration?

Another method of partial purification (can also be used to desalt) that involves a chamber of inert gas that through a pressure gradient pushes everything smaller than a designated size through it onto the filtrate (what stays on top is the retentate)

Chromatography

Separation technique based on differing affinities of solutes who are moving and stationary phase

What is the chromatography process used in this lab?

Gel permeation, also named gel filtration, size exclusion or molecular exclusion

How does gel permeation work?

It uses beads made of sephacryl S-300 HR, an acrylamide (in this lab), that have tiny microscopic pores that can retain and hold smaller proteins. The way it filters through the column, the smallest proteins will be stuck in the beads the longest time, with large ones passing right through the beads and coming out the fastest

What can be obtained from gel permeation?

Apparent molecular weight, as well as also purifying the protein

Why is the weight obtained from gel permeation apparent?

Because the orientation of the protein and other small details can affect how it goes through the column

Void volume (V sub O)

Space in column not taken up by the beads

V sub T

Volume taken up by buffer and gel

V sub B

Volume taken up by beads

How to obtain void volume

V sub T - V sub B

Blue dextran

Can be used to determine V sub B because it is a molecule too large to enter the beads; if this is run, a single peak is obtained, and its peak gives the Ve, which in the case of a molecule that can't enter the beads equals the void volume

Exclusion limit

Weight that can't enter pores of beads

V sub e

Volume of how much has eluted the column up until the peak; for example, every amount up until that point gets added together

What graph is used to determine apparent molecular weight of unknown?

V sub e/V sub O on X axis and log of molecular weight on y axis

Ion exchange chromatography

Type of adsorption chromatography in which molecule to be separated is reversibly bound to a charged functional group of opposite charge

What are the two types of ion exchange chromatography? How do they differ?

Cation and anion exchange chromatography; in cation exchange, the functional groups (ligands) bound to pores of the beads of the column are negative charged, and in anion exchange they are positively charged

How does elution occur in ion exchange chromatography?

After the proteins bind the functional groups in the pores, elution can occur using a salt gradient or pH gradient. Salt gradients uses increasing salt ions that compete for binding in the pores and they elute out of the column depending on how strongly charged they are (ions can outcompete and they leave). pH gradients work by changing the charge on the protein by changing the pH so that the affinity for the beads decreases and they elude out based on which is less strongly charged

Hydrophobic interaction chromatography

Similar to ion exchange but instead of charged, the ligand is hydrophobic; to initially bind the proteins, salt concentration is increased and once all are bound it is gradually decreased and the proteins lose their affinity; the weakest bound and least hydrophobic are eluted first

Chromatofocusing

Similar to ion exchange chromatography, but uses a pH gradient, with the highest pH on the column first and a lower pH added to it to create a gradient (descending pH gradient). Proteins, when added, will then sort according to pH, with proteins that have the highest isoelectric point eluting first

Affinity Chromatography

This chromatography technique is based off of very specific interactions (such as enzyme interactions, antibodies, etc) and that's how it binds to the column. It can then be eluted using competition. Affinity chromatography can also partially unfold and denature a protein

An example of affinity chromatography

AMP and NADH; beta-galactosidase and lactose analog

Why are air bubbles particularly bad for analysis of molecular weight in gel permeation?

The bands on the column get wider as the proteins go down the column, so if an air bubble is presence it can affect the quality of seperation and lead to inconsistent band width changes