Western Blot Help!

[sgragg31]

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.

[Evilynn]

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?

[Warthaug]

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

[mdjww]

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