hello everyone, i performed western bolt about 3 times, but not get pics, during images, the protein bands disappear. Any one help me, where the problems ? thank you
Let's start from the beginning...
1. did you see the proteins transfer onto the blot from the gel?
2. Did you titrate your primary and secondary antibodies? What are the concentrations you are using?
3. Did you stain the gel to make sure that the protein bands are on there?
4. I would try to get you ladder working first.
yes, the protein transferred from gel to blot.
second, i titrated with primary and secondary antibody, and the concentration i used was 1:1000
third, yes, i stained the gel with ECL solution.
I would start with getting your ladder working in the western first, just to eliminate a potential issue with that part of the assay.
So... if you titrated the antibody, then it worked before yes?
Yes, but I think the problem is in antibody concentration or in staining?
Possible explanations could be:
a) the amount of protein is too low.
b) the antibody concentration is too low.
c) your antibody do not worl correctly.
d) the performance is not adequate (aparatures, chamicals, the experimentator).
etc.
If it worked before with the same antibody concentrations, I don't see how if would just stop working. (Now, the only time this happened with another colleague, it was because she changed the antibody concentrations and while it worked once, for whatever reason, it stopped working, and the only way I fixed it was to go back to the original antibody concentration.) Do you have a positive control in there so we can rule out protein concentration as an issue?
The first gel looked like the substrate was not coated evenly, and the second looked like there was too much.
I would still try to get the ladder working first.
Dear Shahid Iqbal
Everyone pointed out reasonable points where you need to see. Before, going into the problem associated with protein of interest, hope endogenous control worked well. since you mentioned transferring of blot worked and antibody titration is already done.
Now, I agree with u that after antibody titration, 1:1000 dilution was considered. Here u can concentrate the antibody and see what is the result as everyone suggested.
Also, u can work transferring condition also, possibly can try combination of different time duration and voltage.
Thank you so much for good suggestions Shen-An Hwang , Saurabh Mandal and Andreas Eisenreich .... I got good result and best image.
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