Protein Gel Electrophoresis

36
Protein Gel Electrophoresis 1.Native PAGE 2.Native Gradient PAGE 3.Urea PAGE 4.SDS PAGE 5.SDS Gradient PAGE 6.IEF 7.2D PAGE 8.Western Blot

description

Protein Gel Electrophoresis. Native PAGE Native Gradient PAGE Urea PAGE SDS PAGE SDS Gradient PAGE IEF 2D PAGE Western Blot. Principle. Proteins move in the electric field. Their relative speed depends on the charge, size, and shape of the protein. From large to small and simple. - PowerPoint PPT Presentation

Transcript of Protein Gel Electrophoresis

Page 1: Protein Gel Electrophoresis

Protein Gel Electrophoresis

1. Native PAGE

2. Native Gradient PAGE

3. Urea PAGE

4. SDS PAGE5. SDS Gradient PAGE

6. IEF

7. 2D PAGE

8. Western Blot

Page 2: Protein Gel Electrophoresis

Principle

Proteins move in the electric field. Their relative speed depends on the charge, size, and shape of the protein

Page 3: Protein Gel Electrophoresis

From large to small and simple

Page 4: Protein Gel Electrophoresis

Protein visualization on gels

Immediately after electrophoresis proteins in the gels are precipitated by either adding alcohol containing solutions or strong acids (e.g. TCA).

Protein are often stained by Coomassie Blue dye or by photography-like treatment with AgNO3 (silver staining)

There are many other stains available (e.g. Stains-all, fluorescence probes etc.)

Page 5: Protein Gel Electrophoresis

Example of silver stained gel

Silver staining is usually 10-100 times more sensitive than Coomassie Blue staining, but it is more complicated.

Faint but still visible bands on this gel contain less than 0.5 ng of protein!

Page 6: Protein Gel Electrophoresis

Native PAGE

Useful for:

1. Examining protein-protein protein-ligand interactions

2. Detecting protein isoforms/conformers

Separates folded proteins and protein-protein or protein-ligand complexes by charge, size, and shape

Page 7: Protein Gel Electrophoresis

Native PAGE examples

In the absence of phospholipids, both twinfilins run as a single sharp band on this gel. PI(4,5)P2 causes twinfilin-1 and twinfilin-2 to move more rapidly toward the anode, indicating a net increase in the negative charge and thus a binding interaction.

Dimerization of KIR2DL1 in the presence of Co2

Vartiainen et al. JBC 2003 Qing R. Fan et al. JBC 2000

Page 8: Protein Gel Electrophoresis

Native gradient PAGE

Separate native proteins by size – proteins stop moving when they reach a sertain gel density (but this may take a very long time ...)

A great technique to study protien oligomerization!

Page 9: Protein Gel Electrophoresis

Native gradient PAGE example

Zavialov et al. Mol. Microbiol. 2002

Native 4-15% gradient PAGE

Page 10: Protein Gel Electrophoresis

Urea PAGE

Separates denatured proteins by size/charge

Typically 6-8 M urea is added into the gel

A great technique to study protein modifications!

Page 11: Protein Gel Electrophoresis

Example of Urea PAGE

Urea PAGE of samples of heat shock protein 25

Zavialov et al. BBA 1998

Page 12: Protein Gel Electrophoresis

SDS PAGE

Due to high density of binding of SDS to proteins, the ratio size/charge is nearly the same for many SDS denatured proteins. Hence proteins are separated only by length of their polypeptide chains (but not by differences in charge).

Great separation. Allows estimation of the size of polypeptide chains

Page 13: Protein Gel Electrophoresis

SDS gradient PAGE

Bands in SDS gradient gel are usually sharper than in homogeneous SDS PAGE

5-20% SDS PAGE

12.5% SDS PAGE

Page 14: Protein Gel Electrophoresis

SDS-PAGESodium Dodecyl Sulfate -

Polyacrylamid Gel Electrophoresis

Page 15: Protein Gel Electrophoresis

Sodium Dodecyl Sulfate• SDS is a common ingredient in detergents• Other names for SDS include laurel sulfate

and sodium laurel sulfate• As a detergent SDS destroys protein

secondary, tertiary and quaternary structure• This makes proteins rod shaped• SDS also sticks to proteins in a ratio of

approximately 1.4 g of SDS for each gram of protein

• Negative charge on the sulfate groups of SDS mask any charge on the protein

Page 16: Protein Gel Electrophoresis

PolarHydrophilic head

Non-polarHydrophobic tail

SDSSodium Dodecyl Sulfate

• Because it is amphipathic, SDS is a potent detergent

H-C-C-C-C-C-C-C-C-C-C-C-C-O-S-O-Na+

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

O

O

C12H25NaO4

S

Page 17: Protein Gel Electrophoresis

SDS and Proteins

SDS

Protein

Page 18: Protein Gel Electrophoresis

SDS and Proteins

In aqueous solutions, SDS polarizes releasing Na+ and retaining a negative charge on the sulfate head

So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains

In the presence of SDS all proteins have uniform shape and charge per unit length

SDS nonpolar chains arrange themselves on proteins and destroy secondary tertiary and quarternary structrure

Thus shape is no longer an issue as the protein SDS complex becomes rod shaped

Page 19: Protein Gel Electrophoresis

Acrylamide

Acrylamide

Polyacrylamide Gels• Polyacrilamide is a polymer made of

acrylamide (C3H5NO) and bis-acrilamide (N,N’-methylene-bis-acrylamide C7H10N2O2)

O

CH

CH2

NH2C

O

CHCH2

NH2C

CH2

bis-Acrylamide

O

CHCH2

NH2C

Acrylamide

Page 20: Protein Gel Electrophoresis

Polyacrylamide Gels

O

CHCH2

NH2C

O

CHCH2

NH2C

SO4-.

• Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate

Page 21: Protein Gel Electrophoresis

Polyacrylamide Gels1. Acrylamide polymerizes in the presence of free

radicals typically supplied by ammonium persulfate

SO4-.

O

CHCH2

NH2C

O

CHCH2

NH2CNH2

O

CHCH2

C

O

CHCH2

NH2C

2.TMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction

Page 22: Protein Gel Electrophoresis

Polyacrylamide Gels• bis-Acrylamide polymerizes along with

acrylamide forming cross-links between acrylamide chainsO

CHCH2

NH2C

O

CHCH2

NH2C

O

CHCH2

NH2CNH2

O

CHCH2

C

O

CHCH2

NH2C

O

CHCH2

NH2C

bis-Acrylamide

O

CH

CH2

NH2C

O

CHCH2

NH2C

CH2

Page 23: Protein Gel Electrophoresis

Polyacrylamide Gels• bis-Acrylamide polymerizes along with

acrylamide forming cross-links between acrylamide chains

Page 24: Protein Gel Electrophoresis

Polyacrylamide Gels• Pore size in gels can be varied by varying the

ratio of acrylamide to bis-acrylamide

Lots of bis-acrylamideLittle bis-acrylamide

Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratio

Page 25: Protein Gel Electrophoresis

1 2 3

SDS-PAGE

Addition of SDS23

1 Protein becomes rod-shaped with uniform charge distribution

Page 26: Protein Gel Electrophoresis

IEF

Separates proteins by their isoelectric points (pI)

Each protein has own pI = pH at which the protein has equal amount of positive and negative charges (the net charge is zero)

Page 27: Protein Gel Electrophoresis

IEFMixtures of ampholytes, small amphoteric molecules with high buffering capacity near their pI, are used to generate the pH gradient.

Positively and negatively charged proteins move to – and +, respectively, until they reach pI.

PI of proteins can be theoretically predicted. Therefore, IEF can also be used for protein identification.

Page 28: Protein Gel Electrophoresis

IEF example

Zavialov A.

IEF 4-6.5 pH gradient

Page 29: Protein Gel Electrophoresis

2D PAGE

Page 30: Protein Gel Electrophoresis

2D PAGE

Lung V79 cells from chinese hamster

Guillermo Senisterra Dept. of Physics-University of Waterloo-Waterloo-Ontario N2L 3G1-Canada

Page 31: Protein Gel Electrophoresis

Proteomics Pathway

Page 32: Protein Gel Electrophoresis

Proteomics Pathway

Page 33: Protein Gel Electrophoresis

Western Blotting (WB)WB is a protein detection technique that combines the separation power of SDS PAGE together with high recognition specificity of antibodies

An antibody against the target protein could be purified from serum of animals (mice, rabbits, goats) immunized with this protein

Alternatively, if protein contains a commonly used tag or epitope, an antibody against the tag/epitope could be purchase from a commercial source (e.g. anti-6 His antibody)

Page 34: Protein Gel Electrophoresis

WB: 4 steps

1. Separation of proteins using SDS PAGE

2. Transfer of the proteins onto e.g. a nitrocellulose membrane (blotting)

3. Immune reactions

4. Visualization

Page 35: Protein Gel Electrophoresis

WB, Step 2: Blotting

Page 36: Protein Gel Electrophoresis

WB, Steps 3-4: Detection