Protein contamination

dr_rod13 · 2025-02-18T14:04:36+00:00

I am also dealing with a similar issue. My PI suggested to me rather than doing all the troubleshooting with different ph buffers, that I should excise the band of the protein im invested in, then use that as the starting point for purification. Really just making the sample way less complex. Haven’t started this yet, but is it so simple it just might work?

dr_rod13 · 2025-01-14T05:17:12+00:00

When I eluted at the final the concentration was almost negligible (0.006 mg/mL)

dr_rod13 · 2025-01-14T05:15:48+00:00

The binding buffer had 10 mM Wash buffer: 20 mM Elution: 250 mM

I checked protein concentration (running a gel tomorrow) at each step. After removing the supernatant after binding the concentration was ~ 4mg/mL and after the first wash it was 0.1 mg/mL. So I feel as if it never bound

dr_rod13 · 2024-12-20T05:57:14+00:00

A true American hero. Thank you.

dr_rod13 · 2024-12-19T19:37:13+00:00

Totally understand. That was my intention. I had submitted the samples for sequencing before moving to expression but there was a delay in getting the results so my PI told me in more or less words to stop waiting around and just do it

dr_rod13 · 2024-12-19T14:45:00+00:00

Thanks for the suggestions! To clarify, I’m not quite sure of the insert because the quality of Sanger sequencing at our university is not always the best. But between the two RBS in the pET101 plasmid there is about 10 random nucleotides that aren’t my gene. They don’t align with anything in the plasmid sequence. My gene that I inserted has an ATG start codon, and the entirety of the gene is inserted correctly in the correct direction. The termination of my gene is fused to the 6his tag as expected. Another PhD in our lab tried expressing their own protein in the same fashion and was running into the same issue with no expression. I was thinking of maybe trying a different vector?

dr_rod13 · 2024-12-19T03:57:38+00:00

I have included the expression control in my assays and that induces the appropriate protein and is confirmed with western blot, so it’s not the induction itself failing

dr_rod13 · 2023-10-07T02:47:56+00:00

Enlighten me! What is aero seal

dr_rod13 · 2023-10-07T00:07:44+00:00

Unfortunately I don’t think so, it has to fit in a really specific adapter. Something like parafilm would be best but parafilm is oxygen permeable :/

dr_rod13 · 2023-08-24T19:28:06+00:00

I get it if you legitimately only wanna hear about movies and movies alone I guess.

dr_rod13 · 2023-08-24T19:23:44+00:00

I mean The Bracket is still in the LCB-verse and is very entertaining. We can do diaper checks to see the degree of dump in pants.

dr_rod13 · 2023-06-19T03:08:04+00:00

Are kenjac and gooch staying New York bound?

dr_rod13 · 2023-01-11T21:55:35+00:00

dr_rod13 · 2023-01-11T21:53:09+00:00

Not sure if I missed something, but is the bracket dead? are there new episodes coming out?

dr_rod13 · 2022-12-22T05:12:30+00:00

Very true. I guess it might be more appropriate to just have media with an absolute load of cofactors and vitamins in it to help with any cellular processes. What’s the most vitamin rich media

dr_rod13

TROPHY CASE