I have performed a western blot with the same samples, but I had problems in my antibodies, so I did once again. However, after the transfer to membrane (ECL nitrocelulose, amersham) only the standard (kaleidoscope) was transfered.
I colored the gel with comassie blue and no bands were there!!!
somebody could suggests what happened?
Detail - despite have problems in antibodies, I could see protein in ponceau and after the digitalization, however it was very low, because of that I performed another experiment. Thus it is the same samples. The only thing I changed was the transference time (30 min bigger, at this time) and the sample buffer, now DTT 6X, and the other was 5X.
Hi Douglas,
If the standard (protein made standard) has been transferred, that means-generally- that the rest of the proteins have also been transferred onto the membrane. To further make sure that all of your proteins have been transferred onto your membrane, you can stain the gel with coomassie blue: if all proteins have been transferred, there shouldn't be any stain on the gel. And you can stain the membraine with ponceau red, the bands should appear.
Based on what you're saying, the proteins seem to be transferred on your membrane... You can troubleshoot for many things here: Go for a higher protein content in the wells (50 microgram, 75, 100 depending on your protein), Increase your primary antibody dilution, Increase your secondary antibody, try another batch of ECL, try an overnight transfer...
Good luck!
If you can see it in Ponceau then the protein has transferred onto your membrane and hence there is no bands with coomassie. I think its the problem with your antibody.
Good luck.
Hi Douglas,
If the standard (protein made standard) has been transferred, that means-generally- that the rest of the proteins have also been transferred onto the membrane. To further make sure that all of your proteins have been transferred onto your membrane, you can stain the gel with coomassie blue: if all proteins have been transferred, there shouldn't be any stain on the gel. And you can stain the membraine with ponceau red, the bands should appear.
Based on what you're saying, the proteins seem to be transferred on your membrane... You can troubleshoot for many things here: Go for a higher protein content in the wells (50 microgram, 75, 100 depending on your protein), Increase your primary antibody dilution, Increase your secondary antibody, try another batch of ECL, try an overnight transfer...
Good luck!
try to stain a housekeeping protein, like actin or GAPDH, so u can understand if it is your antibody which does not work.
Hi, i think it maybe the transfer time that u have used. If there is no bands in gel after comassive blue staining and very poor digitilzation of proteins after ponceau staining in membrane, then it has to deal with the time of transfer. If you are doing dry tansfer then 30 min extra would definetly make the difference, because it would cross the membrane depending on the pore size. And make sure u prepare the sample well. So try once again using the standard time and prepare the sample well. It would solve your problem.
What transferring system are you using? Wet transfer or semi-dry transfer? What is the MW of your protein? If the MW of your protein is high >65KD, then if you are using semi-dry transfer, transfer efficiency will be very low. If you are using wet transfer with very low MW protein, again the efficiency will be low. If everything is fine with your transferring system, I guess the problem most probably is your antibody? Is it monoclonal or Polyclonal? If you are using monoclonal, it could be very specific and may not detect your protein just because it may have been raised against different epitope, check that otherwise try polyclonal antibody against the same protein just to rule out problem of the antibody.
Re transferring. You can transfer overnight at 7V, to ensure full transfer of your proteins. Also trying to increase your protein concentration > 100ug, depending on your protein. Recheck your antibodies - using the correct one ie monoclonal/polyclonal, raised in the the appropriate animal etc. Just a thought :)
there isn't protein in your homogenat or the power of device that you use may insufficient to transfered protein.
Just a thought, but is the pore size of your NC 0.22 µ? With larger pore sizes, I've found that transferred proteins don't bind to the NC as well. Also, are you using 0.1% SDS in the trans-blot buffer as some investigators do? If so, it is very efficient at transferring proteins out of your SDS-PAGE gel but can have the bad effect of causing the proteins to not bind to the NC.
The other posts are all good. I hope your westerns work.
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