Very Strange Western Results

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Jason_Zhang's picture
Very Strange Western Results

I am detecting a protein from a kind of samples. For positive concol, I layer indicated protein in different amounts as 10ng 50ng and 100ng on the gel. But the strange is sometimes I got blot on 50ng and 100ng after developing on film, however, sometimes I didnt get any. The following is the infromation could be useful for your valuable answer.

1) the MW of target protein is 28kD

2) We have two kinds of the purified protein for positive control.
Rat brain sample (we know that it release the target protein, and result is positive.)
Bovine-origined target protein (Results negetive, even in 100ng, but before me other people got positive results, and I also got positive result in 100ng dose before these days.)

3) Primary antibody is from rat.

4) Secondary antibody is Pod-goat anti-rat IgG

5) Sample is prepared by SB with 2-ME and boil for 5 min.

We guess probably this problems happend because the protein we used for positive control come form bovine, well the primary and secondary antibody come from rat. But we compared the struture of the two kinds of protein, 99% same.

Need your answer.

a_wandering_1's picture
In my experience,

In my experience, inconsistency with chemiluminescent Westerns is usually from not having well optimized conditions. Many people have their conditions so far off that they are exposing film for 1-5 seconds - and they're happy with that!? The problem is this makes your results difficult to reproduce accurately, often leads to high background, and rapid loss of signal from the most intense bands.

You need to dilute out your antibodies to get a signal that is stable for at least 30 minutes and requires 1-2 minutes to expose the film (this also gives greater specificity and less background).

Jason_Zhang's picture
Thank you for your kindly

Thank you for your kindly reply.

The developing conditions are OK. I tried 10, 3 mins and overnight incubation with X-ray film. Results same exept stronger and stronger signal in the positive control from Rat brain sample. But the standard target protein from bovine didnt produce any signal.

The background is very clear and beautiful. We have experience on that.

I think the problems should occur from the nature character or the denature system in Sample Buffer, boiling, or eletrophrosis.

Thank you for considering this. I can give you more conditions for the western procedure, if needed.