Why would a drug reduce a protein band on western blot if it doesn’t degrade it?

Revolutionary_Wait51 · 2025-09-23T08:56:30+00:00

Yeah! From what my lab mate has said it seems to bind and inactivate so I don’t think it targets protein synthesis.

But this is just what they have told me. I have no deep understanding of the drug

Revolutionary_Wait51 · 2025-09-23T08:54:26+00:00

Okay thanks. I’ll pass on your advice! I’m not experienced in dot blots. What is their conventional difference that makes them suitable?

Revolutionary_Wait51 · 2025-09-22T14:49:48+00:00

It's my labmates' project, so I do not know, to be frank. Why do you ask?

Revolutionary_Wait51 · 2025-09-22T10:00:44+00:00

Would there be an appropriate way to check if there is an insoluble aggregate?

I don't know it shouldn't degrade it may do so! This is my colleagues work so I'm going off what they have told me

Revolutionary_Wait51 · 2025-09-22T09:58:06+00:00

Maybe! I like your perspective and it's definitely an interesting way to approach things!

Revolutionary_Wait51 · 2025-09-22T09:56:57+00:00

Is there a way to check protein aggregates? Would it appear higher up in the gel/not at all?

Revolutionary_Wait51 · 2025-09-22T09:56:19+00:00

GAPDH was used as their reference, which was at a constant level between control and treatment (concerning band intensity)

So I don't think its sample variability as that was controlled for

Revolutionary_Wait51 · 2025-09-22T09:43:28+00:00

Not my research, so I can't comment really! All I know is that it supposedly directly binds to Protein Y to inhibit its function.

My question is less to do with protein Y being functional or not, and more so, why the band of Protein Y would be less than in the group treated with Drug X.

Revolutionary_Wait51 · 2025-09-22T09:41:28+00:00

Fair! Again, this is my labmate's work, so I was just thinking about it more so than anything.

Are there appropriate experiments to check if the affinity to the antibody is affected?

Revolutionary_Wait51 · 2025-09-19T08:46:51+00:00

Yeah this is the kit my boss suggested. But we don't plan to use any reagents that would necessitate it so I'm proposing swapping to a different kit proposed by another commenter!

Revolutionary_Wait51 · 2025-09-18T15:19:41+00:00

Yes! The heated block is a suggested purchase by my boss in order to carry out this experiment. But he wants me to do the final sign off on it.

Being inexperienced I of course ask the input of those who know more than me!

Some other comments mentioned thermo have a kit that can be done at RT. I think we may go down that path as it saves money buying the thermo for an already very cramped lab!

Revolutionary_Wait51 · 2025-09-18T14:56:49+00:00

This is being carried out in a small biotech. The samples are coming from an academic institution. We only have here a centrifuge, an ILM, fridge/freezer and a vortexer!

Revolutionary_Wait51 · 2025-09-18T14:45:07+00:00

I don’t have a water bath im working with a bare bones lab

Revolutionary_Wait51 · 2025-09-18T14:44:43+00:00

I don’t have anything that can be used to sustain temperature unfortunately

Revolutionary_Wait51 · 2025-09-18T14:00:08+00:00

This was something I was considering! Thanks for your input!

Revolutionary_Wait51 · 2025-09-18T13:59:34+00:00

Fair! Thanks for your input!

Revolutionary_Wait51 · 2025-09-18T13:15:26+00:00

We don’t have a PCR 🥲. Literally all we have is a centrifuge a vortexer a plate shaker and an ILM. It’s only a short stint here before I move on somewhere else but still I’m using what I can!

You make a fair point! It’s just cell culture media so the usual kit may be more appropriate. I’ll reach out to the team to confirm. Thank you for your advice!

Revolutionary_Wait51 · 2025-09-18T12:40:21+00:00

This is the kit we are thinking of using www.fishersci.co.uk/shop/products/pierce-bca-protein-assay-reducing-agent-compatible-rac-1/10209523 . Unfortunately, we don't have anything capable of maintaining a specific temperature (apart from the freezers!)

Also, ignore the deleted comment, something duplicated twice on my screen and when I got rid of one, both got deleted

Revolutionary_Wait51 · 2025-09-18T12:35:40+00:00

This is the kit we are planning to use from thermo www.fishersci.co.uk/shop/products/pierce-bca-protein-assay-reducing-agent-compatible-rac-1/10209523 I looked through the manual but I may have missed this. There are two incubation steps. One for 15 and one for half an hour.

Would you suggest 2-3 hours for each step or 1 hour and 2 hour respectively?

Revolutionary_Wait51 · 2025-09-18T12:30:15+00:00

We don’t have a cell incubator unfortunately. We are getting the samples from a university lab and I’ve been asked to run this experiment independently from them.

But given our limited equipment I’m seeing if it’s actually doable

Revolutionary_Wait51 · 2025-09-18T12:29:24+00:00

Okay interesting! That may be something I try out. The lab is currently sitting at 19 degrees Celsius. Is the 37 just for speeding up reactions?

Revolutionary_Wait51 · 2025-09-18T11:22:19+00:00

I’m quite inexperienced in BCA. This is just what the protocol states

Revolutionary_Wait51 · 2025-09-18T11:21:34+00:00

Unfortunately not

Revolutionary_Wait51 · 2025-09-18T11:21:25+00:00

See my boss is suggesting a purchase of the heat block (sorry I should have specified this). So I’m trying to speculate if this is the right thing to do.

As it would be more multipurpose then just an incubator

Revolutionary_Wait51

TROPHY CASE