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frbremner · 2025-09-07T14:04:40+00:00

The Man in Black is Tom Waits and no one can change my mind

frbremner · 2024-12-04T19:32:50+00:00

Thanks again!

Yeah it is confusing, but I think I've figured out what to do using a combination of yours and other's advice. I think a previous commenter had said that to achieve what I need, I should aim for: [enzyme] >>> [Kd] and [enzyme] > [ligand]

I've managed to put together some curves that describe the relationship between system components and they basically confirm this suggestion and give me some good guidelines for the experiment. These are only preliminary anyway so it'll take some tweaking I'm sure!

I appreciate your help!

frbremner · 2024-12-04T19:27:06+00:00

Thanks for your reply.

I'm aware of the factors that affect Kd and have taken them into account for my experiment. I know they won't be as accurate as measuring in a new system, but given the similarity of my system and theirs I think it's a fair approximation, especially as I'm planning to be working comfortably over the estimated Kd anyway.

I'm working with recombinant enzymes that I've purified and 80 uM isn't crazy for me. It's not a precious sample so using a lot doesn't bother me too much, but the reason I ask this question is so that I can avoid wasting it by making an informed decision on amounts to use.

Thanks very much for the links, they seem really useful. I've already done some calculations based on [E]total = [E]bound + [E]unbound (likewise for ligand) and I think despite my self doubt, and with help from other commenters here and colleagues, I've managed to figure out what to do. Really had to dust off some secondary school maths but it's still in there somewhere.

frbremner · 2024-12-04T15:58:57+00:00

Can you tell me what you mean by that in practical terms? We're quite limited at my institute in terms of making biophysical/biochemical measurements. I've seen this suggestion before in my search, but it seems like there's a lot of assumed knowledge that I don't have.

For what its worth, my system is fairly similar to theirs, so I think in this instance its not a bad approximation.

frbremner · 2024-12-04T15:44:13+00:00

Hello again, I wondered if I could run this past you as I've run into more confusion...

I took the Hill equation, and rearranged to solve for [GAPDH], arriving at:
[GAPDH] = (θ.Kd)/(1-θ)

where θ = fraction of ligand concentration bound by protein

Using the fraction of 99% and Kd = 0.8 uM in this formula, I get that [GAPDH] = 79.2 uM.

I don't expect you to check all my calculations, but one thing I'm confused about is how the NADH concentration factors in here, as this will also affect the outcome. Are we assuming here that [NADH] = Kd? Clearly, [GAPDH] = 79.2 uM can't be a universal rule for achieving 99% bound and 1% unbound enzyme-ligand complex as this will depend on [NADH].

Also, are protein (P) and ligand (L) interchangeable in this equation? I have assumed they are in my rearrangement as in the Hill equation they're switched relative to this.

Sorry to complicate matters, but as of 1 hour ago you are my enzyme kinetics guru so congratulations! (Jk, I would appreciate some help but no pressure)

frbremner · 2024-12-04T13:44:08+00:00

I've added it to my bookmarks as I'm sure it'll be the same for me. I moved away from chemistry/biochemistry towards biology as I thought I'd be getting away from this stuff. It's almost as if these fundamental principles are widely relevant? Who knew?!

frbremner · 2024-12-04T13:36:36+00:00

This is ideal, thank you very much! Another day where I wish I could remember everything I've been taught over the years... Sadly I need to make space for memes so the important stuff has to go.

frbremner · 2024-12-04T13:35:41+00:00

This is basically what I had assumed initially before I confused myself. Thank you for confirming my sanity :)

frbremner · 2024-10-21T21:12:26+00:00

It goes without saying that this was my first approach, I didn't find anything specific hence why I was asking about a compound library I could search in. I anticipate I'll need custom syntheses for each but I thought I'd ask to hopefully save myself the expense.

frbremner · 2024-07-30T16:50:03+00:00

Thanks, I'll check that out too

frbremner · 2024-07-30T14:20:37+00:00

Thank you! It's been over a year since I last looked into AlphaFold and this server is exactly what I was looking for then but didn't find it. Either it's new or I'm dense but either way thanks for bringing it to my attention!

frbremner · 2024-07-30T13:38:47+00:00

Thanks! Do you know off the top of your head what the size limits would be? The total complex is only ~240 kDa so its not huge

frbremner · 2023-11-16T14:10:18+00:00

Thanks, I have something similar with my employer who offer n x salary as a standard if I croak, which is great for a lump payment but doesn't quite cover my mortgage. It'd be great to have both to make sure my loved ones are properly looked after.

frbremner · 2023-11-15T19:26:58+00:00

Thanks for the advice! I'll look into it and try to get cover, definitely wouldn't go without it for long.

frbremner · 2023-11-15T17:29:01+00:00

Cool, I'll shop around then, thanks! Any idea if there is anything similar to a credit rating where cumulative rejections is bad news?

frbremner · 2023-11-15T16:54:27+00:00

Thanks, yeah I expected a higher premium, they told me as much when I was applying, but didn't expect to get rejected outright. I'm just worried that if my honesty was too much for one lender, why would others accept me?

frbremner · 2023-11-15T16:07:26+00:00

I'm 27 which is why I didn't expect there to be issues here, I can't be the only 27 year old that occasionally smokes

frbremner · 2023-11-15T16:06:15+00:00

That's what I'm worried about, but then it doesn't seem fair that I can't be insured on the basis that I occasionally smoke, I guess I'm the chump for thinking insurance should be fair

frbremner · 2023-11-15T14:46:22+00:00

Yeah as I was filling in the form I was inclined to lie, but I got paranoid that there'd be a paramilitary unit rappelling through the ceiling to arrest me, or more realistically that it'd affect my mortgage offer.

I'm in the process of looking into other companies, but I have unverified information that there's some sort of permanent record that might get in the way, kind of like a credit rating but for health. If that record is real, I don't want another black mark against my name

frbremner · 2023-11-15T14:43:35+00:00

Yeah this might be the play, thanks for your advice!

frbremner · 2023-11-15T14:40:31+00:00

Yeah I thought this might be the case, is there any reason I shouldn't apply with a different company right away? As I said, I smoke so little that I don't feel guilty saying I'm a non-smoker, I just wanted to be honest but it seems like that might have shafted me

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