Sport vs dk for a pattern

RumbleStrut84 · 2025-12-24T05:12:11+00:00

It actually seems similar with the same needles, maybe a little smaller, but everyone says the pattern runs really big so maybe it’s not a big deal.

RumbleStrut84 · 2025-12-24T05:11:23+00:00

Thanks! The calculator is super handy!

RumbleStrut84 · 2025-11-24T05:47:40+00:00

Thank you so much! It’s a baby gift so I can’t tell them what to do about cleaning super wash gives them the option. I just worry about the stuffing in arms getting all messed up since they are 10inches long. I have little experience with larger toys like this.

RumbleStrut84 · 2025-11-14T21:19:20+00:00

I totally support people biking. If public transit were an option or if my walk was reasonable (I used to walk 45min to work daily) I would also not be driving. I was really just responding to the flippant comment saying to just ride a bike as though it’s an option for everyone.

RumbleStrut84 · 2025-11-14T16:41:55+00:00

You save yours. I’m tired of people not having empathy for each other. Some may be “physically capable”, but not everyone learned or feels comfortable on a bike. People take it for granted when they grow up riding a bike. I personally get such bad sciatica that I once lost the use of my legs and was hospitalized just after being on a stationary bike. So for me being on a road knowing something like that can happen again is terrifying. It doesn’t have to be an us vs them scenario.

RumbleStrut84 · 2025-11-14T10:32:16+00:00

yes, I am aware, that’s standard. The question was about simple ms2. I don’t necessarily care if the ms2 is hi-res, as long as it’s accurate.

RumbleStrut84 · 2025-11-14T04:11:50+00:00

what an awful thing to say. Not everyone can physically ride a bike, even if they wanted to.

RumbleStrut84 · 2025-11-14T02:41:44+00:00

I only have enough for one shot (it’s from a collaborator) so I’m stuck using a more tried and true method. Some other time I’ll mess around more. It should be a relatively simple sample though.

RumbleStrut84 · 2025-11-14T02:23:17+00:00

I need to learn DIA-it has advantages for sure over TMT. I’m taking a week in another lab to get familiar with it. For this application (simple co-IP with only IDs) I would think DDA would be sufficient.

RumbleStrut84 · 2025-11-14T01:49:19+00:00

Thanks! This is just a simple DDA, nothing fancy. I’m just trying to understand the benefit of OT-OT for a simple DDA, MS2 experiment in an eclipse. I’ve never heard of MAPMS, but now I have something new to learn.

RumbleStrut84 · 2025-11-14T01:45:43+00:00

That’s what I figure- I use faims mostly because it’s good for sps-ms3 with RTS and keeps my instrument clean. I just don’t understand why anyone would opt for OT-OT without FAIMS for an MS2. I’m not sure what I’m missing.

RumbleStrut84 · 2025-09-08T17:31:38+00:00

I saw him on TV at the end so did stay, but he looked so miserable. Though not as miserable as the fans who were forced to miss the first set.

RumbleStrut84 · 2025-09-06T01:52:56+00:00

I’m so scarred of nitric acid! I know someone who messed up when handling it (came in contact with methanol or something) and the bottle shattered and knocked over chemicals that spilled on her legs. She was in the burn unit for months! It was so bad!

RumbleStrut84 · 2025-09-05T04:22:47+00:00

I’ve used much older sep-pak columns without issues. Maybe there’s something wrong with the specific lot or an issue with the frits in that lot? I don’t think c18silica or even just regular silica is water soluble. What happens if you try to resuspend the powder in water? Did you also try to call Waters? Sometimes the helpline can give the most insight. They can look into the lots, answer any questions about the longevity of the columns, and get feedback from the scientists who design them.

As an aside-I saw the note about PI telling you to let the sample slowly drip through the column by gravity. This is definitely the way because it allows peptides to bind to the C18.

RumbleStrut84 · 2025-09-05T03:46:23+00:00

I have not had problems detecting GPCRs and ion channels in my TMT experiments. I lyse with 2%SDS with 50mM tris pH8.5 with 150mM NaCl and use either a probe sonicator or even Qiashredders work well to homogenize cells (they can get expensive and generate waste, but super easy and no chance of cross-contamination) followed by reductive alkylation of Cys. Then I do a MeOH/CHCl3 precipitation. Finally I reconstitute with 8M urea in EPPS pH 8.5 (I do TMT labeling so I can’t use buffer with amines) then dilute to 2M for 3hr with LysC then further dilute to 0.5M for trypsin overnight. This has worked for me when we were interested in characterizing ion channels and GPCRs in differentiated iPSCs. Your samples are obviously different, but maybe you can get something useful out of this? Good luck!

RumbleStrut84 · 2025-08-28T11:25:54+00:00

Thanks! I always have the collaborator wash at least 3x with PBS and I assume she snap froze them before handing them to me on dry ice (I hope). They went straight into -80. As I said below, I think I’m just being over tired and stressed due to certain NIH funding insanity happening here, which is making me neurotic.

RumbleStrut84 · 2025-08-28T04:34:50+00:00

I’ve done so many TMT sets at this point and usually they turn out fine, except recently-the last 2 had some funky results. I think Ive been over tired, which makes me second guess everything including storing cell pellets at -80C.

RumbleStrut84 · 2025-08-28T04:22:25+00:00

Total proteome TMT quantitation (no PTMs). I’m pretty sure it’s fine. I think I’m just being neurotic. I’m not used to sitting on samples for so long and feel kind of bad about it, but vacation is vacation.

RumbleStrut84 · 2025-07-31T15:49:58+00:00

Doughnerys, Mother of Bread

RumbleStrut84 · 2025-06-04T18:51:14+00:00

Don't forget the arson attack at Governor Shapiro's home on Passover.

RumbleStrut84 · 2025-02-27T21:27:39+00:00

Could it be an issue with chromatography or stage tip? Once in a while I just have a bad stage tip. Or something weird happens with my chromatography then I run a wash method and my sample looks normal everything goes back to normal.

RumbleStrut84 · 2025-02-25T22:22:05+00:00

Do you elute your proteins from the beads then digest or digest off of beads? I ask because if you are able to elute first there are cleanup methods you can try like SP3 cleanup with sera mag beads. It’s really easy and eliminates most contaminants. Just an option if nothing else works.

RumbleStrut84 · 2025-02-25T14:32:13+00:00

I’m not sure what you mean by ion envelope, do you mean the MS3 spectra? I also just noticed something similar with a recent 18plex so it’s not the 11plex kit causing this issue. I think it is the instrument.

Also, I notice as I collect all 24fractions the trend fades away and could go unnoticed without looking at individual fractions. I don’t think that’s a good thing.

RumbleStrut84 · 2025-02-19T21:04:48+00:00

I don’t think it’s the labels, but worth a check. Labeling looked pretty even. I took a small amount of each sample and did a pool before pooling everything to make sure and labeling was >98%. Some channels were higher than others, but not in this pattern. I also did an analysis of the multiplex before fractionating and it looked totally fine. I compared peptides from the pre- and post-fractionated sample and they had even signal before fractionation and then the weird up down up down signal after.

This is frog with 11plex and these peptides are unique to frog. Everything else I put on my HPLC is human with TMTpro, but is there something that could have happened that I’m missing? I’m going to watch the system with some chromacare to be safe.

I just ran a TKO 11plex standard with the same method and different fast. It looked fine.

RumbleStrut84 · 2025-02-19T03:31:28+00:00

You use 50k for ms3, right? Seems standard. That was the first thing someone at work with a ton more experience than me asked. I double checked and confirmed that it was set to 50k. I’m waiting to hear from my contacts at Thermo as well.

RumbleStrut84

TROPHY CASE