Hi! I am trying to do Western blot for some cell lysates, but I got stuck in the SDS-PAGE step. ( an image from a sample gel is attached)
After staining the gel with Coomassie blue, it is possible to notice that the bottom half did not develop properly as if the migration halted or messed up, with the vertical streaks looking like the wells (although there was no problems with current/voltage during the run) . If I continue destaining the blue background will fade away, remaining only the samples. But when following with the blot, I did not get the expected band sizes (they are heavier), so the samples did not resolve correctly, although the marker did.
What could be the problem?
The technical details (I already tried with all reagents freshly made in the same run)
- samples were treated with SDS sample buffer with 2-ME, heated 95ºC, 5 min (although this occurs whether or not there are samples in the well).
- The gel is a 12% polyacrylamide gel casted from commercial acrylamide solution
- Running conditions: fridge-cold SDS running buffer (Tris+Glycine+SDS) in the Bio-Rad electrophoresis cell, 200V (but also tried 100V with same results) until the blue dye reaches the bottom of the gel (around 45-50 min when 200V, ~1h50 when 100V).
Any ideas would be helpful! Thank you!
Hi Fábio,
Is the acrylamide also new or is it from a bottle used a long time? Try with another acrylamide to rule out this possibility. Since half of the gel has not a good resolution, is it possible that gel had not been completely adhered to the plates? Or could it be that it has not been in good contact with the running-buffer?
It is better to prepare fresh acrylamide solution in your case and increase sample denaturation time to 10 min instead of 5 min.
Try 8 or 10% gel. Marker separation is not that bad, so focus on the sample preparation. The samples appear to be diluted with less protein; load 5-10ug per lane.
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