I was given a project that requires me to purify a set of transmembrane proteins and it has been an uninterupted stream of pain and suffering for the last 6 months or. Either I get a few micrograms from a few liters of media or the proteins are completely degraded and the bloody non-ionic detergents for membrane protein work are breaking the bank.>oh finally something that looks half-way decent lets do SEC to polish it. Oh no it co-eluted with the detergent micelles which burned the protein from the resulting increase in detergent concentration.For every step forward there are two steps backward.
>>16967988Hey uhhh, buddy this isn't the board for actualy scientists this is the board for retards reposting xitter screenshots and clickbait articles so they can feel better about themselves.Anyway I have no idea how to purify proteins and left the last lab I was in where I attempted it. Are you using an antibody against the specific proteins for the pulldown? Can you make a genetic construct with GFP so you can target that? Do you have enough cash left to just do mass spec or whatever proteomics shit? Can you just nope out and quantify transcript levels and do a more standard assay for theoretical activity of said protein? 6 months is a lot of time to spend on boring shit like this if you're a postdoc/grad student.
>>16967988I have no idea what you said but I’m sorry that happened to you.
>>16967988Your triton x-100, tween 20, and digitonin are breaking the bank? Is your lab in India?>bloodyOh, right. Anyways, I doubt anyone will be able to give you an adequate advice. Molecular biology labwork is kind of like cooking - you got to know your kitchen first.
>>16968029>Can you make a genetic construct with GFP so you can target that? Do you have enough cash left to just do mass spec or whatever proteomics shit? Can you just nope out and quantify transcript levels and do a more standard assay for theoretical activity of said protein?In principle I can do these but the project specifically requires the purified recombinant proteins for some downstream in vitro work (which in and of itself is a whole other can of worms but I'm not even there yet).>6 months is a lot of time to spend on boring shit like this if you're a postdoc/grad student.Oh no its not, conceptually its very engaging and I enjoy reading and thinking about these proteins. Its the practical work that sucks ass.>>16968117>Your triton x-100, tween 20, and digitonin are breaking the bank?No, those are cheap but they either don't solubilize the protein or straight up denature it. I need to use like 0.5-1.0 g of stuff like DDM and LMNG per single purificaion and the cost tends to stack up quickly with repeated failed purifications. I can confidently say I gained reasonable experience as I managed to get some of the protocols to work but it feels as if my PI only focuses on the failed aspects. Despite my gain in knowledge and experience as it pertains to membrane protein biochemistry I would not like to work on such proteins in the future. I miss the days of soluble protein work - this shit is children's play in comparison.
>>16967988you forgot to coom into the beaker.
>>16967988Gut feeling advice ... look up if there is any run-of-the-mill protein which might just block the hydrophobic regions. Assuming your purification or later downstream step can tolerate that as a "contaminant".
>>16968195I presume you are using DDM and LMNG for the micelle size issue? Have you tried using a higher concentration during extraction and then decreasing it 40-50 fold (very little above the cmc). Is your protein eukaryotic or prokaryotic? If the former is true have you tried adding lipids or CHS to stabilize it? Also, to adress the yield problems, have you tried different protease inhibitor mixes before lysis? How fast do you work after pulldown? The protein (especially if it’s transmembrane with buried regions) denature by the hour so how do you manage your workflow? Have you tried changing your expression system (from bacteria to yeast or insect cells for better folding vs your detergent). Wish you luck man!
I know this is going to sound like I am being a complete douchebag, but I think you should fully digest with PLA and PLD, run isoelectric point focusing, and then purify with glycerine, keeping the "purified protein" in glycerine until right before its "downstream usage", at which point you should dilute with ultrapure water 10:1 and TFF at an appropriate cuttof.
That sounds so fucking pointless. The epitome of "bullshit job" if I ever heard one.
