Performing dot blot with cellular extract and western blot anti-GAPDH, we have obtained irregular labeling of our dots with white labeling in the center. How to explain this labeling? 2.5 ul of proteins were directly charged as a spot on dry nitrocellulose membrane before performing Western Blot. Any suggestions?
Hi Veronique
You have too much protein... that's the reason. The spots are saturated and this is because you get a white signal in the center (The film is overburned). So, try to dilute the samples, let's say 1/10 or 1/25 and see what happens.
Hope it helps. Cheers
Paco
Hi Veronique
You have too much protein... that's the reason. The spots are saturated and this is because you get a white signal in the center (The film is overburned). So, try to dilute the samples, let's say 1/10 or 1/25 and see what happens.
Hope it helps. Cheers
Paco
thanks for your excellent answer Mr. Enguita, you solve my problem, I have same problem.
Do you use a suction apparatus? If yes, another possibility would be that the aspiration was too strong. In that case, the drop you put in the wells would have been aspired directly before covering the entire surface of the membrane inside the well. To fix that, just stop the aspiration, drop your sample in the well, be sure that it is covering the whole surface (if not, increase the dilution as suggested above) and then start the suction.
As for me, white labeling in the center is absent of signal in this part. Be tidy when you work with dropper (pipette)
Thanks a lot for your replay. I have just tested loading of serial dilution of protein sample and performed the same WBlot as mentioned above. In fact, I have observed white zone in the center of my drops in the lowest concentrated load but not in the higher one. So I think that it's not a problem of too high protein concentration (with saturation of the chemical detection) but a problem of sample loading. I don't use aspiration loading but only manual loading. If you have other suggestion, it will be very helpful. Thanks
Since, as you said, it is not a problem with the sample concentration, perhaps you should try using suction. Connecting the dot blot apparatus to a vaccum pump is easy and usually results in efficient transfer. Try to do it as suggested above- by Nicolas.
I agree that it could be a problem with the loading, I have had that when using the classical yellow tips, then I have chaged to those filtered of 20 or 10 ul and let the drop form at the end to the tip just before touching the membrane so the drop is just spread. Other option is to load you sample the most slowly you could, so the drop became smaller in diameter and also you avoid the white center that can result from a concentration of the protein in the borders of the drop when in the membrane. I was charging 2-4 ul of lysate.
Good luck!
Dear Veronique
Hope you solve your problem by using the diluent. If not the linkage with UV crosslinker(120mj) is maybe usefull. Good luck
Adding to Patricia´s suggestion you can use a Hamilton pipette of the proper volume with a blunt end needle (to avoid rupturing the membrane) as the drops are much smaller. And wait for complete absorption between drops.
I too have had the same problem! I solved it by using a larger volume (100ul), three sheets of blotting paper, the nitro rehydrated in water before being used, and a reduced suction power!
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