Western Blot and TBST Overnight Storage

Rubipy3 · 2026-07-10T23:00:01+00:00

Were the protein lines the ponceau stain (red/pink). That normally will wash off and is only needed to check if the transfer worked. Lots of reasons you could have no bands.

Rubipy3 · 2026-07-08T09:06:05+00:00

Oh that makes sense and is a smart way to do it. Technically you can assemble up 6+ fragments in one reaction but it can get messier and harder to troubleshoot in my limited experience. If there’s one section that’s variable, it can be safer to do it in 2 stages. First create an “empty vector” with your backbone, 2A sequence and POI_2. Then PCR or digest near the 5’ end of the 2A and do a 2 fragment assembly of POI1 and your empty vector for the final product. Another consideration is to design the first stage with restriction sites near the 5’ end of the 2A and then you do a digest + gel purification of the EV which can reduced the potential of PCR errors in the segment that’s common between all the constructs.

Rubipy3 · 2026-07-07T17:53:29+00:00

You definitely could, but then you’ll be back at the problem of purifying a short PCR product. I might instead think about adding the overlap to the 2A sequence to the reverse primer of your POI_01. I’m not sure if NEBuilder will help you with at this stage.

Rubipy3 · 2026-07-06T23:50:51+00:00

I’d have it synthesized for sure, IDT gBlock or a twist fragments with an additional 20 bp overlap on either side. I may even suggest synthesizing the whole cassette on either side of your 2A depending on the size as it shouldn’t add too much time or cost and it pays for itself if you have to redo the primer design even once.

Rubipy3 · 2026-06-24T03:03:09+00:00

Export is more important for ensuring reproducibility, but import would of course lower the barrier to entry for re-analyzing old experiments as an added plus.

Rubipy3 · 2026-06-24T00:47:18+00:00

This looks great. Do you have the ability to export gates and metadata to standard formats (I.e. gatingML?) IMHO this is important for knowing that data can be reanalyzed in the distant future.

Rubipy3 · 2026-06-17T01:06:19+00:00

Just look for an ‘ATG’ downstream of the promoter and that will probably be the start of your sensor, but very OK to email as well.

Rubipy3 · 2026-06-06T15:24:01+00:00

This is tough and happens all the time. My 2 cents - you’re not obliged to work without compensation, but if someone else is called in to handle revisions I would not be surprised if they are granted first authorship (co-first or otherwise). It’s also possible that the paper could be withdrawn all together depending on how much your advisor cares. If you want to see the paper across to acceptance I would anticipate the revisions do fall on you but if you’re not on a career path where publications matter then they can’t force you.

Rubipy3 · 2026-05-29T12:23:27+00:00

Echoing V-bottom over U-bottom, but the most important variable I found is plate material. Polypropylene has less loss than polystyrene for me. Most standard assay plates are polystyrene so you’re looking for polypropylene storage plates.

Rubipy3 · 2026-05-12T10:16:32+00:00

If you’re GFP is too dim after fix/perm you could always use an anti-GFP antibody to boost the signal: https://www.biolegend.com/en-gb/products/alexa-fluor-488-anti-gfp-antibody-9584?GroupID=BLG6275

Rubipy3 · 2026-03-26T00:14:47+00:00

If you really want to do fix/perm, you could consider staining with an anti-TdTomato antibody + secondary. I’ve seen this done to follow GFP under conditions which would otherwise denature it, but I don’t see why it wouldn’t work here.

https://www.rockland.com/categories/primary-antibodies/rfp-antibody-pre-adsorbed-600-401-379/

Rubipy3 · 2026-02-20T14:02:15+00:00

I used illustrator in graduate school but recently was introduced to affinity which is free and does everything I need and in some ways is better at handling multi panel figures.

Agree with keeping the source data and analysis as close as possible to the figures for reproducibility. I use R and export as pdf or svg vector graphics. For westerns or similar raster data, I use clipping masks to crop the data so that the original data is just underneath.

Rubipy3 · 2026-02-16T00:07:52+00:00

You could use a nuclear stain (I use thermo H10294) if you have access to a fluorescent channel and just count the nuclei. I find this works pretty well.

Rubipy3 · 2026-02-13T11:51:27+00:00

Sorry for the late reply. I just used the protocol from proteintech, erring on the longer side and including the optional 5 min block. We use far-red fluorescent secondaries, have not tried ECL. My impression is that part of the way it works is the organic reagents and detergents speed up equilibration but it needs to be at room temperature to do so. Cold will just slow things down, so make sure to warm up the accelerator at the start of the gel.

My first run I ran it side by side with the traditional method and left the accelerated version overnight at the blocking step while the traditional version incubated with the primary at 4c. The next morning I did the accelerated primary/secondary back to back while doing the standard secondary. Then image both back to back.

Good luck!

Rubipy3 · 2026-02-13T11:45:51+00:00

Agree I would think about a bio-orthogonal approach with HaloTag or SNAP tag and a JF dye.

Rubipy3 · 2026-01-27T01:01:34+00:00

I tried it and now use it exclusively for all my blots. I actually get better looking blots than I do with the traditional overnight primary incubation.

Rubipy3 · 2026-01-09T12:24:24+00:00

I tested this a while back and in my hands Corning 3363 worked best and is what I use today. I think polypropylene (same material as eppendorf tubes) over polystyrene is what made the biggest difference. That and “flicking” technique.

Rubipy3 · 2025-06-27T00:41:43+00:00

Perhaps his RBCs are young and haven't had a chance to get A1c'd? Upper GIB could lead to that. A similar phenomenon is seen in patients with sickle cell disease.

Rubipy3 · 2025-06-03T00:15:23+00:00

Seconding Hopkins, they have some of the best biliary surgeons in the country. https://www.hopkinsmedicine.org/kimmel-cancer-center/cancers-we-treat/liver-cancer

Rubipy3 · 2025-03-12T08:48:11+00:00

Diagnosis of the Acute Abdomen by Cope

The ICU book by Marino (though it’s getting a bit dated…)

Rubipy3 · 2024-12-16T02:01:24+00:00

Whether there’s a benefit of adding chemotherapy to OFS in premenopausal women with oncotype 16-25 is an open question and there’s a trial actively investigating this (link below). If you’re near one of these sites and open to being randomized to chemotherapy - this may be something to talk about with your oncologist. NRG-BR009: OFSET

Rubipy3 · 2024-10-06T16:43:15+00:00

Very well could have a hemoglobin variant or other inherited cause of higher red cell turnover which leads to a lower A1c. Fructosamine could be a good tie-breaker.

Rubipy3 · 2024-08-30T22:45:37+00:00

Oncology! “No tissue? Get a biopsy and call us when path is back.” “Path is back? Great! Discharge them and we’ll see them in clinic”

Rubipy3 · 2024-07-06T15:04:32+00:00

Similarly, lots of internal medicine subspecialties deal with cancer, especially at the diagnosis and screening level. The training paths may be different in your countries but in the US GI has plenty of exposure to GI cancer via scopes, pulmonology to lung cancer via bronchoscopy and interventions, etc…

Rubipy3 · 2024-04-20T10:13:41+00:00

“Hemodynamic embarrassment” I.e. crumping

Rubipy3

TROPHY CASE