According to the color or the loading buffer and the protein ladder, the pull-down progressed successfully. And I also get right signals of beta-actin (lower ones), but another primary antibody (Na+/K+ ATPase mouse mab) only stains the top area of each strip, which is not the protein's right place. I used RIPA buffer and boiled the samples for 10 min. What would be the problem that may cause this situation?
Try using a Blotting Grade blocker to significantly lower the background noise
It seems that there is still binding to other large proteins. Did you add some mercaptoethanol or Dithiothreatol to your solution before loading it onto the gel?
Ankit Naik So you think its the background noise? well I had another gel processed today and had the same problem, I'm now pretty sure its nonspecific binding to all proteins and especially easier to bind bigger proteins. Since in actin situation, the primary antibodies were HRP conjugated, so I didn't use any secondaries. But to others, I did - so I think the problem maybe the improper use (or improper dilution rate) of the secondaries/primaries. Any suggestions?
Mohammad Parvaiz Farshori
Thank you for you suggestion. I've found the problem which is proteins larger than 50 kd could not be successfully pulled down & separated. I'm using the reducing loading buffer from thermo fisher, and boiled the mixture for 10 min then spin down at 17,000 g for 5 min. I don't know why this keeps happening. I'll try IEF if get a chance.
It appears that some of the protein stuck in the well, it did not even enter the separating gel. There are plenty of possible reasons for that: too much protein; conflict between buffering system of electrophoresis and sample, pH, salt concentration, detergent, etc.; accidental loading of beads into the well.
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