Hi all,

I need help. I was trying to detect 2 different proteins in cell lysates via Western. I bought 2 Rabbit polyclonal Antibodies (2 companies). In both cases, I used Pierce 2ary antibodies, Donkey anti-Rabbit AP and HRP H&L (tried both detection systems).

Nothing.

As a test, I diluted both the primary Antibodies 1:10 and 1:1000 and ran them out directly on a gel and transferred them to NC filters. I then probed these filters with the 2 secondary antibodies (1:1000) and developed them by their methods (AP and HRP).

One of the two 1:10 diluted primary antibodies was detected by both methods, although only 1 band instead of H & L.

The other was not detected.

The 1:1000 dilution was also not detected.

I doubt the protein of interest will be concentrated enough in my lysates to be detected.

Where to go from here?

Thanks,

Ed

it is hard to say without running a positive control

usually, antibodies supplied at ~1ug/ul, assume you have a 20ul loading, 1/10 dilution results a 2ul protein, which should be detectable by both generic and chemiluminescence (and actually, you will be able to see bands with CBB stain).

I do not know your detection methods, but 1/1000 will probably not detectable by generic reaction

I will suggest to concentrate your lysates by precipitation

If you think that your protein of interest is too dilute in your lysates, there are a number of options you can take to optimize your Westerns.
1) What was your method for detection? Pierce has a product called SuperSignal West Femto Substrate for ECL analysis (http://www.piercenet.com/Products/Browse.cfm?fldID=01041106) that can detect as little as 1 femptogram of protein in the lysate. It is likely that this would be sufficient for even an extremely poorly expressed protein or if there is a question of your primary antibody being poorly sensitive, this reagent may help you. I have had luck with it in the past detecting poorly expressed proteins.
2) You may also want to check the efficacy of your primary antibody. Have you tried producing your protein of interest in vitro (rabbit reticulocyte or wheat germ system) to get large amounts of it and testing the antibody on your more purified protein?
3) Sometimes a simple immunoprecipiation can also be used to "purify" your protein of interest, making it more visible on a Western blot.
4) If your protein is nuclear and associates with nuclear structures, generating nuclear extracts also concentrates your protein, making it more visible on a Western.

Hope this helps.

I used 2 different methods. 1) Alk Phos 2ary with BCIP/NBT substrate and 2) HRP with Pierce's SuperSignal Pico substrate, which may not have been sensative enough. The cellular lysates may not have enough target protein, so a method of concentrating them, like IP, could be the answer.

Thanks,

Ed