7-8. techniques in biochemistry

importance of pure proteins

studying enzyme function, drug development, structural analysis, and post-translational modifications

You can fractionate proteins through

centrifugation

Salting out is based on

solubility of proteins varies with salt concentration

salting in

adding salt until a protein dissolves in water

salting out

keep adding salt until protein precipitates

Successive additions of ammonium

sulfate, with centrifugation at each

step

can separate proteins according

to their relative solubility

Dialysis

can be used to remove salt, or

change the buffer

Gel-Filtration/Size Exclusion Chromatography- what comes out first

bigger molecules come out first since they don’t get stuck in pores of column

Ion-exchange Chromatography

separation of proteins by charge

In an anion exchange column, what elutes first

positive proteins do not bind with column, so they flow through first

In an anion exchange column, after cations have eluted, how do you get the anions off the column

high salt buffer which displaces the ions with Cl ANIONS

IN an anion exchange the matrix is

positively charged beads that attract anions.

IN an cation exchange the matrix is

negatively charged beads that attract cations.

In an cation exchange column, after anions have eluted, how do you get the cations off the column

high salt buffer which displaces the cations with Na cations.

Does a protein at its pI

still bind to an ion-

exchange column?

yes because the protein is neutral, but it may have charged residues to interact with column

Affinity Chromatography

separates protein using specific binding to antibody or resin

When carrying out affinity chromatography, how would you get the protein off the column if it is bound to antibodies

add a competitor OR change ionic environment (like low pH)

ow can you separate charged molecules

electrophoresis

What matrix in electrophoresis is used for proteins

Polyacrylamide gel

SDS and Mercaptoethanol

used to prepare proteins for separation based on size by denaturing proteins and reducing disulfide bonds.

A solution of proteins treated with SDS and Mercaptoethanol all be

monomers, unfolded with a net negative charge

During PolyAcrylamide protein Gel Electrophoresis (PAGE), proteins move in what direction after SDS

towards the positive electrode

During PolyAcrylamide protein Gel Electrophoresis (PAGE), where are biggest proteins on gel

located near the top

Isoelectric Focusing

Separate Proteins with Respect to their pI

When carrying out isoelectric focusing, acidic proteins will be

migrate toward the positively charged lower pH

When carrying out isoelectric focusing, basic proteins will be

migrate toward the negatively charged higher pH

low pI

more acidic side chain —> more negative charge —> goes to positively charged region of gel

high pI

more basic side chains —> more positive charge —> goes to negatively charged region of gel

2-D gel electrophoresis

can be used to resolve changes in protein expression and protein modifications

Methods to detect proteins separated by gel electrophoresis include:

1. Proteins stains

2. Radioactive labels

3. Western blots and antibodies

Edman degradation.

sequentially removes one residue at a time from the N terminal of a peptide to determine the amino acid sequence.

Strategy for sequencing an entire protein:

Cleave the entire protein into smaller fragments, using

a protease or a chemical cleavage method then sequence peptides

Cyanogen bromide

cleaves carboxyl side of methionine residues

Trypsin

cleaves carboxyl side of lysine and arginine residues

Chymotrypsin

cleaves carboxyl side of phenylalanine, tyrosine, and tryptophan, leucine, and methionine

Mass spectrometry

highly sensitive technique to determine a protein’s identity using purified sample from gel band

Alcohol dehydrogenase assay

If you mix alcohol dehydrogenase with

ethanol and NAD+, you should see an

increase in absorbance at 340nm, then you can measure specific and total activity

Alcohol dehydrogenase

catalyzes the oxidation of ethanol to acetaldehyde (NAD+ is reduced to NADH)

Specific Activity

calculated to Assess the Purity and Quality for a Protein of Interest

how to calculate the specific activity

divide total activity by total protein

WHat two methods are used to determine tertiary structure of proteins

X-ray crystallography and NMR spectroscopy

X-ray crystallography

used a perfect crystal to study the arrangement of atoms in a protein by diffracting X-rays.

NMR

can be done on protein samples in aqueous solution

Immunoglobulin G (IgG) antibody

is a Y- shaped hetero-tetrameric protein

Fab

antigen binding fragment of an antibody that binds to specific antigens.

Sc (Fc)

single chain stem of antibody

VL

is the variable region of the light chain in an antibody that contributes to antigen specificity. (TIP of the Y)

Cl

is the constant region of the light chain in an antibody that provides structural support (base of the Y structure) and is vital for the antibody's stability.

scFv

single chain variable fragments (using just the tip of the Y) can be made from genetic engineering

Epitope

specific part of antigen recognized by antibody

Specifically for protein antigens, the size of epitope is

typically 6 aa residues

polyclonal

antibodies that are derived from multiple B cell lineages, recognizing different epitopes on the same antigen.

Antibody

producing cells

can be cloned to

make

monoclonal antibodies that recognize a single epitope

Western blotting

used to identify proteins

separated by SDS-PAGE

Western blots are incubated with

primary antibodies that specifically bind to the target protein, then secondary antibody will induce color change

ELISAs

are assays used to detect and quantify proteins, antibodies, or hormones in a sample by using enzyme-linked antibodies.