I am doing lots of Western Blots and never had any major issues with it. However, I have been struggling to get a nice transfer for a protein of 150 kDa. In the attached figure you see lysates of different cells expressing that protein (the lower band is a 120 kDa isoform). I'm expecting the bands to be (more or less) identical in intensity. My loading controls and other proteins look perfect, so it's not unequal loading. I have repeated the experiment at least 5-6 times and always get that pattern. It's always a different cell line showing very high or low intensity bands, so I don't think it reflects real major expression differences.
I genuinely don't understand why that happens, in particular because I didn't think 150 kDa would be such a high molecular weight for a transfer. My loading control is 100 kDa and looks good.
I have tried many things, including nitrocellulose vs PVDF membranes, constant current vs constant voltage, increasing transfer time, changing detergent concentration in my washing buffer etc. I am doing wet transfer only.
After transfer, I do a Ponceau stain and the overall transfer looks good, but unfortunately I cannot see the area above 120 kDa because I think I have very low amounts of proteins. Still, it's weird that I only can't see the higher region.
My samples are RPE1 cell lysates coming from a 10 cm cell culture dish. Due to their morphology, their density is not that high and I only get very small cell pellets. I did a test transfer with HEK cell lysates yesterday and my bands for the respective protein looked wonderful (could also see the bands in the Ponceau very well). So, here the transfer worked nicely. I think that I had at least a 10-fold higher amount of cells.
So, it somehow seems like my problem is a mixture of having very low amounts of cells/proteins and decreased transfer efficiency due to higher molecular weight. Is that possible? I can't really explain why every lane seems to have a very different and individual transfer efficiency.
My current settings are: PVDF membrane 0.45 um pores, transfer at 100 V for 2h and
I would suggest you try a control transfer where you use the same amount of the same lysate in each lane. That way you can rule out sample differences and be certain that it really is a transfer issue (or not).
you can trying increasing methanol in the transfer buffer. If you have been using 10% then increase to 20%. Also I would transfer overnight at 30-100mA.
Also, your expectation for similar identical bands might not be correct and you are actually not doing anything wrong. Comparing across cell lines might show differences in your protein of interest. Or your quantification of protein for each line is not equivalent if you have to use different buffers to lyse.
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