Also, for some reason when I make homogenates of mouse skin tissue, they give roughly equal protein estimations between genotypes but one is consistently 3-5x more concentrated than the other. This led me to run 4 15-well gels yesterday and 4 10-well gels today with single-microliter changes in sample volume to try to get one pair that has equal actin bands. But since the lipid content is so high (after 3 rounds of spinning and skimming fat off the top and a 100K trip in the ultracentrifuge), 75% of the samples contorted and/or precipitated somewhere in the stacking gel. I just don't get it.
- Current Location:UMB Lab
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..I failed.
PS time for a new icon, this one is starting to give me the booboojeebies.
I like screencapture and outright theft for my icon supply.
Precipitating in the gel?????? I'll assume the gel has SDS in it as well as the running/loading buffer? Lastly, double check the pH of the Tris in the resolving gel layer. Precipitation problems are often related to pH there.
If regular lysate is loaded, the lipids bubble out of the wells and even when ultra'd, they leave visible white lipids at the bottom of the wells when they migrate. actin on one genotype is basically fine, on the other it's just a faint cloud or smear 20 kDa above normal and looks as if I loaded the same amount in all wells. Normal 10% SDS-PAGE, works like a charm for cell lysates. I use up and remake 100mL bottles of resolving gel tris every 10-14 days (I run a lot of gels), I made this one 2 days ago, dead on 6.8.
I use a detergent-heavy RIPA usually so I made one w/o any detergents, same lipid problems. Liver, kidney, etc. is no problem but skin is just a sonofabitch to do evenly/correctly. It confounds Bradford, Laemmli, and me in one fell swoop.