3.1.2 Pe Electrophoretic Techniques

Front 1

How is electrophoretic techniques different from chromotagraphic

Back 1

Electrophoretic techniques are for accurate and precise analysis of proteins rather than simple seperation. Electrophoretic techniques require denaturation of the protein.

Electrophoretic techniques are used to monitor the progress of purification and check for purity.

Front 2

What are the 2 steps of preperation for electrophoresis?

Back 2

Breaking of H bonds and VDW by sodium dodecyl sulphate detergent to form micellular bubbles surrounding hydrophobic regions
beta-mercaptoethanol breaks disulphide bridges to form nascent polypeptide

Front 3

What are the 2 dyes used in SDS page electrophoresis and what are the resolutions?

Back 3

Coomassie Blue stain - 10ng
Silver stain - 1ng

Front 4

What charge is SDS and how is that relevant to how SDS works?

Back 4

SDS is negative charge - moves through solid gel matrix towards anode.

Front 5

Why don't we use silver staining as much?

Back 5

Takes a long time to stain, expensive and use of nasty chemicals

Front 6

Why is western blot sometimes used after an SDS-page electrophoresis?

Back 6

Can't differentiate and seperate specific protein if there are lots of proteins of similar mass

Front 7

What are the 6 steps for western blot?

Back 7

Gel Electrophoresis

Transfer proteins to membrane (nitrocellulose) following gel electrophoresis using electroblotting

Blocking - ensure the membrane and other proteins have zero affinity with the probe (anitbody/dye) so wash with buffer and nonspecific protein (e.g. BSA) to reduce the noise

Probe the blot with specific antibodies

Probe blot with second antibody linked to an enzyme - this amplifies the signal

Add enzyme substrate - chemiluminescent signal

Front 8

What is isoelectric focusing? How does it seperate the proteins?
(3)

Back 8

electrophoresis based on a pH gradient.

Solution of proteins added to a column containing immobilised pH gradient (IPG) gels

Proteins move through the gel until they have reached pI

Front 9

What is 2D gel electrophoresis?

Back 9

Separation based on both charge and mass - use of isoelectric focussing followed by SDS page gel electrophoresis

Front 10

How do you concentrate the protein after electrophoresis?

Back 10

Use a molecular weight cut off filter in a tube - put proteins on top of filter in tube and centrifuge
Solute and buffer (unwanted) filter through the filter and is discarded

Front 11

What are the 2 reasons for concentrating a protein after using an electrophoretic technique?

Back 11

To do different assays
For repeatability

Front 12

What is the concentration required for crytallisation?

For EM?

Back 12

10 mg/ml for Crystallisation

0.1mg/ml for EM

Front 13

Lowry Assay is a way of measuring total _ _ _ _ _ _ _ concentration & relies on the _ _ _ _ _ _ _ _ _ of _ _^_ _ ions in Lowry reagent (phospho_ _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ acid) by peptide bonds, causing the _ _ _ _ _ _ _ _ _ of aromatic residues resulting in the _ _ _ _ _ _ _ _ _ of _ _ ^_ _ to _ _ ^ _ _

The colour changes are then detected in a visible light spectro photometer

Back 13

Protein

Chelation
Cu2+
phosphomolybdic-tungstic acid
oxidation
reduction
Cu2+
Cu+ or Cu1+ (same thing)

Front 14

Reduction of Lowry reagent results in a solution that has a _ _ _ _ colour, and absorbance can be measured at the _ _ _nm wavelength
The concentration of protein in solution can then be found by looking at a standard curve.

Back 14

Blue

760

Front 15

What are the possible assays once you have purified the protein and checked it? (3)

Back 15

Amino acid composition

Primary sequence of protein

Structure of Protein

Front 16

What are the 3 steps involved in degrading a protein non-sequentially find the amino acid composition?

Back 16

Dissolve protein in 6M HCl and put solution in an evacuated ampoule (a glass capsule)

Heat to 378-383K for 24 hours

Separate using cation exchange column

Front 17

What is the downside of harsh hydrolysis of proteins? How is this resolved?

Back 17

Trp, Ser, Tyr, Thr destroyed. by hydrolysis
Tyr, Thr, Ser protected by addition of phenol or thiol

Trp can be detected by A280

Front 18

What is the cation exchange column usually made of? What properties enables it to separate amino acids?

Back 18

Made of sulphonated polystyrenes

Separates amino acids in 2 ways:

1. negatively charged

2. The polystyrene is hydrophobic

Front 19

How do you find out the amount of amino acid in a protein after electrophoresis and purification without using fluorescence?

Back 19

Ninhydrin reaction

Add ninhydrin and heat to 373K

To form purple compound

Front 20

What is an alternative to ninhydrin?

Back 20

Phthalaldehyde and Beta mercaptoethanol - reacts with AA to give fluorescent complex (isoindole derivative of amino acid)

Front 21

What are the 2 steps (each step has 1 requirement) for breaking down a disulphide linkage, and what is the product?

Back 21

Reductive alkylation

Using beta-mercaptoethanol

Requires excess thiol (drive equilibrium forward)

Acetylation using iodoacetate

Requires iodoacetic acid excess

Produces acetylated cysteine residues

Front 22

How to separate the following using ion exchange chromatography?

V-S-L-A-G
D-E-G-K-P-R

Back 22

Both have net 0 charge at physiological pH

Run the chromatography at pH 1.9

At that pH V-S-L-A-G is +1

and

D-E-G-K-P-R is +3

as COO- becomes neutralised