No protein in cytoplasmic extract - (Jan/06/2012 )
I am trying to run SDS-PAGE Western Blot using cytoplasmic and nuclear extract from NIH: Ovcar 3 cells (ovarian cancer). When I am running the gel I see that the cytoplasmic extracts are running irregularly along the gel some moving faster while some slower. The bands look thicker and seems to be diffusing. However the nuclear extract seems to be perfectly alright. I could see my proteins of interest in the nuclear extract. I can see small bands ( very faint) in my cytoplasmic extract but strangely I cannot see any actin band.
I fear that the proteins in the cytoplasmic extract are getting degraded by some means.
I would really appreciate any kind of help or suggestion.
check the salt content of the extract - it would appear that there is probably too much salt for good electrophoresis. You may need to dialyse the samples before running.