Hi Bei, I have fought this problem myself several times. In addition to some of the suggestions from Tim, here are some suggestions for additives:
As you mentioned Tween20 and Triton X-100 are very good for solubilizing difficult insoluble proteins. Those two are usually my first two weapons against insolubility. NP-40 (this is a detergent, but as far as I know it's not available commercially anymore, but you can buy "NP-40 substitute" from several companies) Glycerol (already mentioned) Xylitol or Sorbitol (which also work well as cryoprotectants if your protein/crystals like(s) them) Trehalose (+ or - extra phosphate in the buffer) 0.1M Arginine Also, it's best to start with low concentrations of these additives and increase them as needed. As for working with detergents, if your protein is soluble and not a membrane protein, you want to be well below the CMC. Like 50% of the CMC, max. The idea is that you want the soluble detergent molecules to interact with the sticky parts of your protein, and if the detergent molecules are forming micelles they aren't doing their job very effectively. Additionally, and I know this sounds a little insane - I was a skeptic at first, but others in my lab have had success with sonicating solutions of aggregated protein at very low intensity, very briefly. If your protein is reasonably stable but forms aggregates, I think this could work. (Like I said, I didn't beleive it at first, but CD scans of the sonicated protein show that it's still folded properly, and crystal structures have been solved from these samples.) One last thing to mention, when you add some of these additives to your buffers they will become contaminated very easily. Those stubborn little microorganisms that manage to contaminate buffers as simple as tris and salt will LOVE to eat up some of these sugars, amino acids, and even detergents, so be extra careful about autoclaving bottles, filtering buffers, etc. Good luck, as you are no doubt finding out this can be a difficult and frustrating problem. Mike Thompson ----- Original Message ----- From: "joybeiyang" <joybeiy...@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, September 7, 2010 12:30:04 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] off topic-aggregation of proteins Hi everyone, sorry for this none CCP4 question, but I am working on some tough proteins which easily got aggregated after purification, I am thinking of using some detergents rightnow, however, I am a newbie in this field, could you please give me some advice on the usage of detergents? here is some basic information about the protein: 1. This protein is as big as 90KD. 2. The productivity of the protein is very low, and the homogeneity of the protein is not good. Most of the proteins come out of the column at void volumn, and the rest of the proteins forms 3-4 peaks, and each of them features low peak height. 3. I have already tried different truncates of the protein and homologous proteins from other species, up to now the protein on my hand is the best of them. 4. I have stocks of the following detergents:n-Octyl-b-D, n-Decyl-b-D, FOS-Choline-12, n-Dodecyl-b-D, Cymal-5, CHAPS, Tween20, Triton-X100. my question is: 1. would you please recommend some other detergents to try? 2. should I try different concentration of the detergent and how? right now I just tried the concentration equals the CMC, some of the detergents do improve the homogeneity of the protein, however, once I concentrate the protein for crystallization, the detergent get concentrated too, and as you know that is very bad for crystallization. 3. would you please recommend me some literature to resort to (about the same situation as mine or about the use of detergents)? 4. any other suggestions or comments about how to improve the quality of the protein. Your suggestions and comments will help me a lot and will be highly appreciated. Many thanks to all of you! Bei 2010-09-07 joybeiyang -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
