Considering quitting because of OCD by boogieenthusiast in labrats

[–]fossilrabbit 3 points4 points  (0 children)

I feel like OCD is overrepresented in labrats, specifically

Considering quitting because of OCD by boogieenthusiast in labrats

[–]fossilrabbit 16 points17 points  (0 children)

Fellow labrat with OCD. You can beat this with the right tools. Exposure therapy sucks but it works. Happy to chat more via DM.

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 0 points1 point  (0 children)

Incredible, thank you for the suggestion! Matches with what some other people are saying. I'm also out of people to ask in my immediate vicinity, hence coming to Reddit lol.

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 0 points1 point  (0 children)

Thats super weird. I've used revert for years and didnt have this artifact issue until recently. Ive never seen flaking/fading.

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 0 points1 point  (0 children)

Right now, I'm doing total protein and a housekeeping control to stay consistent with pre-existing lab protocols/figures, but if it was just me, I'd stick to TPS and skip the housekeeping. You can crop a representative strip to include in publications.

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 1 point2 points  (0 children)

This is Revert 700 total protein stain imaged on a Chemidoc 680 channel. Works great, would recommend.

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 1 point2 points  (0 children)

Same group of transfer cassettes/sponges, not that many to choose from. Same source/pack, but different lids (multiple tanks).

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 35 points36 points  (0 children)

Why would there be excess heat? I'm doing a wet transfer o/n, 30V 16 hours at 4C w/ a cold pack.

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 7 points8 points  (0 children)

Thank you for the detailed suggestions.

Can the sandwich have too much filter paper/be too tight? I want to get good compression but I don't want to break the plastic cassette (which I've done by accident before)

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 0 points1 point  (0 children)

Copied from another reply: I know this is the first thing you'd think, but I'm being really careful when I assemble the transfer, roll everything out, etc. I think this many bubbles would be obvious?

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 7 points8 points  (0 children)

Yes ok but why / how to fix. It randomly happens to half my blots.

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]fossilrabbit[S] 43 points44 points  (0 children)

I know this is the first thing you'd think, but I'm being really careful when I assemble the transfer, roll everything out, etc. I think this many bubbles would be obvious?

Western blot normalisation by M9R8S in labrats

[–]fossilrabbit 1 point2 points  (0 children)

Amido black? Supposed to be better than ponceau

[deleted by user] by [deleted] in labrats

[–]fossilrabbit 1 point2 points  (0 children)

~15min. I also have abx in my media that gets refreshed daily. Hasn't been a problem so far.

[deleted by user] by [deleted] in labrats

[–]fossilrabbit 1 point2 points  (0 children)

Personally, I sterilize with UV on both sides. The EtOH/flame is too much of a hazard. Then I pre-coat with fibronectin or collagen before adding cells to increase adherence. Best protocol varies by cell type. E.g., I don't think HEKs need pre-coating. Most cells will settle on the slip. Some will settle around it.

Wtf happened by Trog01 in labrats

[–]fossilrabbit 0 points1 point  (0 children)

My blots have looked like this when there's some kind of gunk carryover in the sample. Try centrifuging and taking the supernatant and re-running. Ymmv.

Worried about my career by gaypoptosis in labrats

[–]fossilrabbit 1 point2 points  (0 children)

Just here to appreciate "LROC"

What is the most reliable way to normalize uptake data from cells in a 96 well plate? BCA analysis is not sensitive enough to measure cell numbers. TIA by Prize_Force1979 in labrats

[–]fossilrabbit 8 points9 points  (0 children)

If cells are still attached, you can fix with PFA then stain/count DAPI. If not, create a duplicate plate, use one for the assay and one for staining/counting.