How to choose an antibody? - for western or other applications (Jan/22/2006 )

I am interested in studying one protein so i would like to buy an Ab that allows me to detect the endogenous protein, at least , on WB.
What I would like to learn with this is what kind of considerations one must have when purchasing an Ab? How to decide which Ab is more suitable? What caracteristics are the most important?

From the below available Ab, what is the best antibody option to buy?


1)
purified mouse monoclonal (Ab raised using purified GST fusion protein) ;
Specificity: Human:positive (other sp.not tested);
Applications ELISA, Western blotting (WB), others not tested.

2)
purified rabbit polyclonal antybody (Ab generated from rabbits immunized with a recombinant protein encoding the human full-lenght protein.;
Specificity: Human:positive (other sp.not tested);
Applications ELISA, WB, Immunohistochemistry (IHC), others not tested.

3)
purified rabbit polyclonal antibody (Ab generated from rabbits immunized with a KLH conjugated sintetic peptide (10-30 a.a. lenght) at the N-terminal of first 50a.a. of human protein;
Specificity: Human:positive (other sp.not tested);
Applications: ELISA, WB, IHC, others not tested.

4)
purified rabbit polyclonal antybody (Ab generated from rabbits immunized with a KLH conjugated sintetic peptide (10-30 a.a. lenght) at the C-terminal of last 50a.a. of human protein;
Specificity: Human:positive (other sp.not tested);
Applications: ELISA, IHC, others not tested.

5)
purified mouse monoclonal antibody;
reactivity:mouse, rat, human, dog, chicken;
isotype IgG2a;
applications: IF,IHC,IP,WB.


- Well, from all of these I believe the last option is the best because it enables me to use it for so many thecniches (WB, IF, IHC and IP)
therefore I can study not only protein expression (WB)and localization (IF and IHC) but also thesting interation with other proteins (IP).
Although what worries me is that in product description is not shown a picture to exemplify the specificity and signal.
Should I write to the company and ask for more information on this product?

- Why I am worried? Because for Ab 1) and 3) I can see a WB picture where it shows that Ab doesn't recognize a single band but 4 and 6 bands, respectively. the Ab 2) recognized only 1 band in WB.

- if you know about interesting websites for beguinners in immunology please let me know.

Thank you very much

Macedo, if you look on the pdf or whatever that pops up on most 'product info' or description pages, there should be a couple of references. It is useful to download the references and see pictures of actual data obtained using that product, and then you can also get an idea from the materials&methods how the protocol might run.

another useful tip: when we are trying to decide which antibody to buy, we look to see what others in our field are having success with...again, it involves going to the literature and seeing what has worked for others. most well-written papers will give at least a vendor for their antibody, and hopefully will tell you what dilution was used in their application.

ciao
A