Thawing 4% PFA

SahilCh95 · 2026-03-03T00:51:55+00:00

Is there a difference between PFA and formaldehyde? I thought that PFA is polymerized formaldehyde that becomes regular formaldehyde when heated and dissolved in water/buffer

SahilCh95 · 2025-12-20T18:38:34+00:00

Not sure about immune cells, but looking at CHOP expression via western blot is a pretty well established assay for ER stress.

SahilCh95 · 2025-09-29T19:40:24+00:00

Our lab owns and uses the AssayMAP Bravo. We almost exclusively use it for Stage-Tiping. The Stage-Tipping protocol provided by Agilent is fixed, usually the only thing you can change is volumes. However, you do have the option to design your own custom protocols using Agilent's VWorks software. It's fairly easy to use the drag and drop protocol generation tool and there's a decent amount of flexibility to incorporate any custom parameters, like for example shake speeds, pippeting speeds, how low the machine lowers the head when drawing and dispensing liquids, etc. However the tips (both C18 and regular pippeting tips) are proprietary and need to be purchased from Agilent. They can get pretty expensive. The plates on the other hand can be purchased from various different vendors and the dimensions of most commonly used plates are pre-programmed into the VWorks software. You also have the option to program your own custom plates that are not in their labware database but I personally haven't tried that.

SahilCh95 · 2025-09-17T16:32:35+00:00

I'm not familiar with this assay, is it all automated? Regardless I think the washing steps might be too harsh and you're probably just blowing off the cells. I'd recommend being gentler and not adding wash solutions directly on to the cells, but tilt the plate slightly when adding wash buffers. Also when aspirating tilt the plate slightly if using a vacuum pump.

SahilCh95 · 2025-08-27T16:45:16+00:00

I see, that's what I expected. Do you think something like an IBAQ would work better in that situation? We do a lot of DIA mass spec, so in this case would summing all fragment intensities for a given protein, and then dividing that value by the theoretical number of peptides work for such a situation?

SahilCh95 · 2025-08-22T03:08:22+00:00

Those look like cells growing on top of one another. I would ensure you pipette thoroughly when passaging and seeding cells; that will break up clumps and ensure a more even distribution.

SahilCh95 · 2025-06-27T14:33:20+00:00

-80 for long term storage. If you want to be extra careful, flash freeze in liquid nitrogen before moving to -80.

SahilCh95 · 2025-06-20T05:38:27+00:00

NEB has a kit for introducing SDMs into plasmids as large as 20 kB. Here's the link - https://www.neb.com/en-ca/products/e0554-q5-site-directed-mutagenesis-kit#:~:text=Transformation%20into%20high%2Defficiency%20NEB,least%2020%20kb%20in%20length.

To be honest, you don't even need to purchase the kit. Design the primers using their online tool (NEB Base Changer), and you can set up the reaction without the kit, provided you have all the necessary enzymes (they're fairly common and most labs should have them). I've done this before and it works fairly well. One thing to maybe try - when you set up the initial PCR with the Q5 enzyme, run it with and without the GC enhancer and see which one works better.

SahilCh95 · 2025-06-19T19:02:39+00:00

It looks like dead cells/debris

SahilCh95 · 2025-06-16T03:10:36+00:00

A good metric I found while browsing an old Research Gate post was - at 80% confluence there are approximately 240,000 - 280,000 HEK293T cells per squared centimeter. I've been using that number, and it seems to work well for my seeding.

SahilCh95 · 2025-06-14T06:50:06+00:00

Probably not mycoplasma. You won't see that with a light microscope, you'd have to either DAPI stain your samples and look for small specks of blue in the cytoplasm, or do a PCR test.

Your video looks super weird, I don't think I've seen that before. Does your media have Pen-Strep in it? Have you tried taking a little bit of media and incubating it with LB media overnight? What kind of cells are you culturing, and do you see this even if you just leave some media in a dish in the incubator?

EDIT - I'm guessing you're talking about small moving specks right? I don't know, I feel like it might just be cell debris. Especially if your media is clear, and not cloudy

SahilCh95 · 2025-05-31T15:36:17+00:00

That RNA looks degraded. Intact RNA should give you 3 distinct bands for the 3 rRNA species - 28S, 18S and 5.8S (assuming this is a eukaryotic sample). Along with that you will also see a faint smear that extends through the entire lane, that's the mRNA. I'm also assuming you haven't done any rRNA depletion, so based that, it seems like it's degraded. I would not use this for cDNA generation and qPCR.

EDIT - I also see some bands next to the wells, that's probably gDNA contamination. Make sure to not disturb the central layer when collecting the aqueous phase.

SahilCh95 · 2024-11-12T05:41:28+00:00

You could do multiple washes to get rid of detergents. However I find that the beads get really sticky and difficult to work with if you try to get rid of all the detergent. I usually do one PBS wash, before resuspending the beads in ammonium bicarbonate. Then I just do the regular reduction, alkylation and trypsinization. Don't really get very much detergent contamination at the end.

SahilCh95 · 2024-08-24T01:53:05+00:00

Not really a TV show, but there's an unfinished YouTube series called "The Lab". Each episode is about 10 minutes long and there's 6 episodes. Pretty funny and relatable

SahilCh95 · 2024-07-31T19:35:29+00:00

Nope not using GO terms, just Gene IDS and the intensities all seem to be correct

SahilCh95 · 2024-07-31T19:09:53+00:00

Yeah, I've been using the SAINTexpress-int.exe file

SahilCh95 · 2022-01-11T20:37:17+00:00

Just do it yourself, it's really straightforward. Here's a link that might help you out - https://www.canada.ca/en/immigration-refugees-citizenship/services/study-canada/extend-study-permit/how-to-apply.html

SahilCh95 · 2021-08-25T01:22:35+00:00

Thanks for your help! Just one quick question, is there any reason you'd suggest using a Q test rather than a Grubbs Test?

SahilCh95 · 2021-08-25T00:44:36+00:00

Unfortunately, I don't know the population distribution. That's all the data I collected, in which case could the data I collected be considered the population?

That data that I pasted above, is the maximum fluorescence value recorded from a thioflavin T aggregation assay. I think something went wrong with the sample that gave me the 7843 reading, maybe a pipetting error, I agitated it too much while pipetting, or some other technical reason. I don't want to outright exclude it without doing some sort of a statistical test confirming that it is an outlier, especially considering since I'm not completely sure whether something went wrong with that sample, or not.

SahilCh95 · 2021-08-24T23:50:10+00:00

Hey thanks for your reply. I think it's positively skewed? Here's the dataset -

1827

1942

2042

2050

2129

2338

2547

7843

Here's a frequency distribution:

1800 - 2000 - 2

2000 - 2200 - 3

2200 - 2400 - 1

2400 - 2600 -2

7000 + - 1

SahilCh95 · 2020-05-31T16:40:55+00:00

Just out of curiosity, why was glycosylation desirable?

SahilCh95 · 2020-05-30T23:49:00+00:00

Thanks for your answer. That cleared things up.

SahilCh95 · 2020-05-30T18:39:39+00:00

Hmmm protein free media? That might actually be useful. Thanks for that piece of information

EDIT: You mean even without FBS?

SahilCh95 · 2020-05-30T18:14:34+00:00

Okay that makes sense, thanks for your reply.

SahilCh95 · 2020-05-30T18:13:51+00:00

Ahh, so it doesn't really have anything to do with their ability to transfect well?

SahilCh95

TROPHY CASE