electrophoretic methods: purification and...

Protein purification and characterization

Exercise #8 - Electrophoretic methods

Protein Purification & Characterization

Electrophoresis

• Characterize (degradation, MW)

• Quantify*

• Determine purity of sample

• Compare proteins from different sources

• Required step for western blotting*

Gels

Why standard protein electrophoresis

uses Acrylamide and not Agarose?

Native & denatured PAGE

• Acrylamide/Bisacrylamide

– Inert & thermo-stable

– Control over cross-linking

– Transparent

– Synthetic (reproducible)

• Glycine

– Source for trailing ion (slow mobility)

• Tris-HCl

– Source for leading ion (high mobility)

?האם אנו נקבל בהכרח הפרדה על פי גודל

Gel separation efficiency

15% 5%10%

The length of the gel counts!

(Remember Chromatography?)

Gradient gel is composed of multiple layes of varying % PA

Native gel

Native gel is w/o SDS – how will it run?

How will protein complex will behave under such condition?

In native gels, protein run along the gel according to their total

charge.

What will happen to a mixture of proteins which are

loaded on such gel? How will they run?

The advantage of native gel is the keeping of the fold.

How can we use this advantage?

Dimerization of

KIR2DL1 in the

presence of Co2

Qing R. Fan et al. JBC 2000

Troubleshooting “ugly” gels

Smearing

Membrane associated proteinsOverload + uneven casting of gel

Don’t overload!

(use 20ug-40ug/lane max)

Copyright David R. Caprette 2005

Troubleshooting “ugly” gels

Streaking

Copyright David R. Caprette 2005

Local precipitation

Troubleshooting “ugly” gels

Gradual fading bands

Copyright David R. Caprette 2005

O.N incubation in DDW

Troubleshooting “ugly” gels

Skewed run

Copyright David R. Caprette 2005

Spacers not in place – interrupt the electric field

Troubleshooting “ugly” gels

Bizzare run

Copyright David R. Caprette 2005

Lifting the top hatch (electrode) off while running…

Isoelectric focusing (IEF)

2D gels

Gels are one of the key resources for monitoring the purification process:

• The Degree of protein purification.

•Fractions

• Is the protein present in several fractions?

• Before and after check

• Protein stability over time