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fatty fatty

I celebrated my one year anniversary today by discovering every single plate and dish of cells I have teeming with contamination.

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.

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( 8 comments — Leave a comment )
(Deleted comment)
floyd_mephit
May. 6th, 2008 12:59 am (UTC)
I tried to think of something funny to say about fursuiting helping with microbiology.









..I failed.
silversledge
May. 6th, 2008 12:06 am (UTC)
nerd
floyd_mephit
May. 6th, 2008 12:57 am (UTC)
Oh go condition some operants!


PS time for a new icon, this one is starting to give me the booboojeebies.
silversledge
May. 6th, 2008 02:00 am (UTC)
let me go see what I can dig up
silversledge
May. 6th, 2008 02:15 am (UTC)
Ah, that's better.
floyd_mephit
May. 6th, 2008 02:22 pm (UTC)
creepy, but less creepy...

I like screencapture and outright theft for my icon supply.
skorzy
May. 6th, 2008 12:15 am (UTC)
How are you titrating the volumes of protein samples? If you're making microliter changes from lane to lane, you're very likely experiencing big time pipetting error in those quantities. Probably do better to do 10% changes by serial titration, and do at least two loads for each point.

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.
floyd_mephit
May. 6th, 2008 12:54 am (UTC)
I made master loading buffer samples and took aliquots from those. I had problem with only one genotype, the other loaded on the same gels had even stepwise incrementing. Cell culture samples were same. Just the one knockout has superhigh lipid content (although both require 100k spins to become translucent). My pipettes were just recal'd and loaded with P-20 from highest to lowest vol so I don't suspect much pipetting error, but even if so, it shouldn't cause one set to precipitate and the other not to in the same gel; I don't even care what the volume is as long as it gives several different increasing amounts so as to let me 'pick a winner'.

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.
( 8 comments — Leave a comment )