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Western Blot Help!

Western Blot Help! - Biology Forum

Western Blot Help! - Biology Forums. Ask questions and discuss the study of Biology. If you have Biology questions from your homework ask them here!


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Old 01-23-2010, 08:30 AM
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Default Western Blot Help!



Alright, so I'm trying to learn the methodology and theory behind Western Blot, but I have this question that's been nagging me, and I can't seem to find a specific answer to it.

Why is gel electrophoresis necessary in the Western Blot process?

I know how it separates proteins based on their Molecular Weight, but what is the point when you've got an antibody tag that's going to bind to a specific protein. I mean, my reasoning is that if that antibody interacts with the homogenate of a particular cell line, then isn't that a basis for gene expression and the presence of a specific protein. Somebody please help.
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Old 01-27-2010, 09:29 PM
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Default Re: Western Blot Help!

If all you wanted to know was if the protein of interest is present, you can do that in much simpler ways than a western. Westerns can show you the size of the protein in addition to displaying different levels of expression with regards to a control.

Does this help?
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Old 01-28-2010, 01:06 PM
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Default Re: Western Blot Help!

The gel electrophoresis step is critical, for many reasons:

1) It concentrates your protein in a small area, which can improve detection of rare proteins compared to other detection techniques such as dot-blots.

2) Its far more quantitative than simpler methods. You can compare protein expression levels with WB; you cannot with dot blots, IF, etc.

3) Despite what is usually taught (and shown on product data sheets), antibodies can, and often do, cross-react with other proteins. This is obvious in a western, as you'll end up with multiple bands, while with other methods you'll simply see a signal and as such you'll have no idea if what you're seeing is your protein-of-interest, or a cross-reaction.

4) Western blotting can detect some forms of post-translational modification. Many PTM's cause changes in a proteins weight; for example, some heavily phosphorylated proteins will form a double band on a gel - a lower-weight unphosphorylated band and a higher-weight phosphorylated one.

There are lots of other reasons why the electrophoresis step can be benificial, that's simply the short list...

Bryan
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Old 03-17-2010, 04:56 AM
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Default Re: Western Blot Help!

i have checked it’s really great [Only registered users see links. ]

Last edited by mdjww; 03-17-2010 at 05:00 AM.
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