Western

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Wed Aug 2 03:07:13 EST 2000


Hi Kun,
Here are the basics for western blots.

1. you need a primary antibody against the protein of your choice.
Either, buy a commercial one if you work on a common protein, or use a
home-made antiserum. Most commonly used antibodies are either from
rabbit (polyclonal antiserum) or mouse (monoclonal antibody). There is
no general preference for one or the other, it simply has to work.... :)

2. you need a secondary antibody that recognizes the primary antibody
and has some activity you can detect. Commonly used are anti-rabbit or
anti-mouse antibodies coupled to peroxidase, alkaline phosphatase,
fluorochromes or gold particles. For western blots, peroxidase-coupled
antibodies are most often used nowadays because of their high
sensitivity. Because secondary antibodies are usually purchased, the
companies make lots of it and therefore use big animals, sheep, donkey,
horse... There is no preference for either. 

3. you need a detection system. Chemiluminiscence is most commonly used
nowadays. Colorimetric detection (eg. BCIP/NBT) is also common but is a
bit old-fashioned and has several drawbacks. 


In your case, use the rabbit-anti-your-protein primary in combination
with a peroxidase-coupled anti-rabbit secondary antibody (e.g. from
Sigma) and ECL detection (Amersham). (no affiliations to either company!)


In addition, a low signal may have many reasons:

* low amount of antigen (low-abundance proteins like transcription factors)
* low antibody quality, or too high dilution of the antibody
* low detection sensitivity (use enhanced chemiluminiscence ECL, or a
even more sensitive kit from Pierce)
* too short incubations (no shorter than 1h for primary, 0.5h for secondary)


Frank



PS: Is there no-one around in your department who can give you a good
introduction into western blotting?




Kun Qian wrote:
> 
> Hi, thanks a lot for answering my last question on protein electrophoresis,
> I check all your recommendation and found that there was something wrong
> with teh pH of the buffer, I adjust the pH and everything is fine now.
> But here is another problem i encountered these days, I am doing western and
> get no band or dim band. I am wondering if the primary and secondaty
> antibody combination is wrong or not, can you check that for me?
> The sample I used was from rat heart, the primary antibody is rabbit anti
> rat, and the secondary antibody is sheep and rabbit IgG (H&L).
> sorry for bothering you all with this kind of silly question, your answers
> are greatly appreciated.
> thanks
> Kun Qian






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