Protein seperation techniques clarification

[Whiteshoes]

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For gel electrophoresis - larger proteins move faster through the medium.
For Western blot - Larger proteins move slower through the medium.

The question that had me wondering about this is on this post but no clarification was made.
http://forums.studentdoctor.net/showthread.php?t=538893

 

[Raiden2012]

I thought smaller proteins moved faster for gel electrophoresis?

Based on wikipedia, it seems to agree at least lol

 

[deleted103644]

This depends completely on what kind of electrophoresis you're talking about.

There are two variables for electrophoresis that determine migration rate - charge and "size".

For a standard protein gel electrophoresis, you denature the proteins and add negative charges to them with SDS (a detergent).

For these types of gels, only the length of the peptide chain really matters - longer proteins migrate more slowly, just like in DNA electrophoresis.

You can also run non-denatured proteins, in which case conformation matters (larger effective radius --> slower), or even run a gel without adding extra negative ions to the proteins, so some may run forward, backwards, or not at all depending on pH of the gel and pKa of the protein.

Also, western blot refers to what you do after you run the gel: you transfer the proteins from the gel to nitrocellulose and label it. This can be done with many types of techniques, including 2D gel electrophoresis and other fancier things.

 

[Whiteshoes]

I thought smaller proteins moved faster for gel electrophoresis?

Based on wikipedia, it seems to agree at least lol

yeah i thought the same thing until i took TBR 3 and that question came up..

here's a website that says the opposite of what you're saying lol
http://www.sigmaaldrich.com/life-sc...matography/gel-filtration-chromatography.html


EDIT: that link says gel filtration which i guess is different from gel electrophoresis. So i guess I'd like to clarify that Larger proteins move faster in gel filtration but slower in gel electrophoresis & western blot

 

[Raiden2012]

yeah i thought the same thing until i took TBR 3 and that question came up..

here's a website that says the opposite of what you're saying lol
http://www.sigmaaldrich.com/life-sc...matography/gel-filtration-chromatography.html

I believe the chromatography you mentioned in the link utilizes Size exclusion chromatography which isn't exactly the same. For sec, the smaller particles get trapped in the smaller holes whereas in electrophoresis they travel through faster

 

[StIGMA]

electrophoresis and western blot are the same technique. A western blot is electrophoresis followed by probing with an antibody. Small proteins migrate faster.

Chromatography is different. When based on size, large proteins are not retained by the beads and hence migrate faster.

 

[Raiden2012]

http://en.wikipedia.org/wiki/Size-exclusion_chromatography

This might help

 

[deleted103644]

Size exclusion chromatography is a completely different beast.

Do not confuse with gel electrophoresis.

 

[Whiteshoes]

electrophoresis and western blot are the same technique. A western blot is electrophoresis followed by probing with an antibody. Small proteins migrate faster.

Chromatography is different. When based on size, large proteins are not retained by the beads and hence migrate faster.

http://en.wikipedia.org/wiki/Size-exclusion_chromatography

This might help

Thanks..