Hi Alsagaby,
Some questions for you.
1. Do you have a positive control in your blot?
2. Are you sure your samples have the target protein in a detectable level?
3. Do you know if the quality of antibodies (primary and secondary) is ok?
4. Anything is new in these blots, eg. sample buffers, transfer buffer, wash buffer, etc.?
Sometimes, even if you see the protein marker transferred onto the membrane, it doesn't mean that the protein of your interest was well-resolved and transferred, because the protein marker is well-prepared and ready for running electrophoresis, while some proteins may require special conditions (urea, reducing agent, up to 8% SDS, etc). I heard that my colleaque uses the protein marker directly without adding sample buffer, but I like to use the same sample buffer as for other samples on the same gel, otherwise, it may have strong distortion effect which I experienced many times before. Moreover, I think different sample buffers should have effects on the mobility of the same protein. So, adding same sample buffer to all samples on the same gel should give a better estimation of the protein sizes. I don't know which way is correct. Any slightly change in your sample buffer may affect the solubility/denaturing of some tricky proteins, which means
that you can't exclude the possibility that your target protein become insoluble or not well denatured when you are running the gel. So, the protein marker may not tell you enough information.
Hope you will find out the problem very soon and let us know what it is.
Cheers,
Stanley
________________________________
From: "Alsagaby, Suliman" <salsag from essex.ac.uk>
To: methods from magpie.bio.indiana.edu
Sent: Sunday, June 14, 2009 2:16:37 AM
Subject: Western Blotting problems
Hello
I did western blot twice and every time I have the same problem in which
the PVDF membrane does not contain any protein although it contains
protein marker. The bands were visualized by staining the gel. So it
seems to me that the bands did not transfer to the membrane. I verified
the transfer duration: one for 12h at 30V and the other one was for 1h
at 100V.
I am great full if any body can help me and direct me to the right
protocol if I am doing wrong one.
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