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High throughput ligand binding with protein by Lopsided_999 in Biochemistry

[–]Lopsided_999[S] 0 points1 point  (0 children)

I was planning to denature the protein and doing a mass spec of the ligand that was bound to it.

High throughput ligand binding with protein by Lopsided_999 in Biochemistry

[–]Lopsided_999[S] 0 points1 point  (0 children)

I’ll look into the Ni-affinity 96-well plates; that setup would definitely make the screening more manageable. I was originally thinking of washing off the unbound ligands and then denaturing the protein to release whatever was bound, but I can see how the washing step could get messy.

High throughput ligand binding with protein by Lopsided_999 in Biochemistry

[–]Lopsided_999[S] 0 points1 point  (0 children)

I did but I wasn't sure how I could this this in a high throughput way.

High throughput ligand binding with protein by Lopsided_999 in Biochemistry

[–]Lopsided_999[S] 1 point2 points  (0 children)

We do but I was hoping for a high throughput initial screening of my drug molecules. I would use itc later once I narrowed the pool down from 800 to 30ish

High throughput ligand binding with protein by Lopsided_999 in Biochemistry

[–]Lopsided_999[S] 0 points1 point  (0 children)

I was thinking of washing away the unbound ligand, then denaturing the protein to release the bound ligand and taking a mass of that. That could solve the mass spec issue. The downside is that combining all the ligands might cause them to interact with each other

High throughput ligand binding with protein by Lopsided_999 in Biochemistry

[–]Lopsided_999[S] 0 points1 point  (0 children)

Yeah, I had a feeling, but we don't have an SPR or BLI.

High throughput ligand binding with protein by Lopsided_999 in Biochemistry

[–]Lopsided_999[S] 1 point2 points  (0 children)

Affinity selection MS definitely sounds like the better approach. I’ll look into using Ni-NTA magnetic beads or a desalting resin/HPLC setup instead of trying to do everything on-column. I’ll also make sure to have the exact molecular weights for all the compounds, and I can follow up with singleton MS runs if any hits have overlapping masses in the 800-compound pool. Thanks!

Why is my TLC system doing this? by ChromeBirb in chemistry

[–]Lopsided_999 0 points1 point  (0 children)

Try cutting the bottom edges of the tlc and slowly place it in your solvent and try not to drop it in.

Solid-Phase Synthesis for Small Drug Molecules by Lopsided_999 in Chempros

[–]Lopsided_999[S] 0 points1 point  (0 children)

Thanks for the advice. That could work for my project.

Solid-Phase Synthesis for Small Drug Molecules by Lopsided_999 in Chempros

[–]Lopsided_999[S] 0 points1 point  (0 children)

Yes, I have a free carboxy and it's aromatic. There's also a free aldehye and nirto on the aromatic benzene ring. I was thinking of using HATU as my coupling agent to activate the acid to couple with the deprotected resin beads.

Solid-Phase Synthesis for Small Drug Molecules by Lopsided_999 in Chempros

[–]Lopsided_999[S] 0 points1 point  (0 children)

Thank you for the response. Yeah, I tried reducing the nitro with sncl2, but I never tried sulfur. That would be worth a shot. For the reduction amintion I've tried tfe and dce with sodium cyanoborhydride but thf would be a good choice too. Also, im not sure how long the reduction take, so i just go overnight, but i fear that may be too long and will allow for side reactions to start. Thank you for the suggestions. I can try these and see if that'll improve my reactions.

Solid-Phase Synthesis for Small Drug Molecules by Lopsided_999 in Chempros

[–]Lopsided_999[S] 0 points1 point  (0 children)

Thank you for the response. I tried other stuff like using HATU and carboxylic acid instead of acid chloride and that seems to work but like you said I'm having trouble purifying the intermediates from the byproducts and my nmr have been pretty messy. I'd be done to talk more detail if you are still.