Anita, To answer a lot of your questions, you need to provide some more information about your protein, at least some of its biophysical characteristics (size, oligomeric state, an integral membrane protein, etc.). Just because you are using detergents does not mean all your problems are detergent related. Also you need to provide a bit more information about the conditions where you see "crystals," particularly about the presence of divalent cations and/or phosphate and the detergent concentrations.
Lots of things will crystallize: contaminating proteins, small molecules, and (although not as often) detergents. The fact that you see "crystals" under conditions with quite different detergents suggests that the crystals are not detergents (unless you are very unlucky). For example, C10E6 is a well characterized and studied detergent that has no apparent crystalline phase in aqueous solution. Thus, your hexagonal crystals are interesting. My own bias is that I don't trust what the Izit dye suggests, because its partitioning is not exclusively dependent on protein. If your conditions don't have ammonium ions or free amines (i.e., no Tris buffer, ammonium sulfate, etc.), try adding a microliter droplet of glutaraldehyde next to your protein drop. The glutaraldehyde will vapor diffuse into the protein droplet and protein crystals will become fixed via the lysines. They won't dissolve in buffer; they will also turn a bit yellow from the Schiff's bases being formed. Works great on membrane proteins. It is possible that you can get different crystal habits using different detergents, but providing more information about the conditions of the hits and the detergent concentrations would make it easier to give you suggestions. Were you actually using 5% DDM in the drop or using a 5% DDM stock? With many precipitants, high detergent concentrations results in substantial phase separation. Do you see this? When evaluated suspected "hits" from screens on a membrane protein, I first think of inorganic compounds, particularly the dreaded struvite (NH4MgPO4), which occurs at very low [PO4], but high ammonium and magnesium concentrations. I next consider the quality of the protein I am producing and the reproducibility of the purification scheme. Sometimes a "too pure" protein prep won't crystallize, while a "less pure" prep does fine. Contaminating proteins, particularly those that copurify with Ni-chelation resins (see Bolanos-Garcia and Davies, Biochimica et Biophysica Acta 1760 (2006) 1304–1313 and Structural Genomics Consortium NATURE METHODS 5(2) 135, 2008) can also crystallize at concentrations below 1 mg/mL. Your protein could appear to be 95% pure, but but a 1-2% contaminant could be crystallizing, albeit poorly. A strategy might be 1) to repeat with different protein preps to demonstrate reproducibility and 2) to tweak some of your conditions to explore a wider environmental space. Then evaluate whether your protein preparation protocol needs optimization. While a problem, don't focus to much on the detergent a sole source of your problems. Cheers, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** On May 6, 2012, at 7:45 AM, anita p wrote: > Hi All, > Thanks for your advices. I tried staining with izit dye, please have a look > at the attached image. The granules are all stained. > The detergent used here is Chaps (as an additive to crystallization condition) > Does it look like protein ?? > If yes, please do advice on methods to improve this. > > > I found granules in some other conditions as well and they didn't stain with > izit. So probably they were of detergent... Plz suggest > Thanks in advance for suggestions > regards > Anita > > > On Fri, May 4, 2012 at 11:43 PM, John K Lee <kw...@msg.ucsf.edu> wrote: > Hi Anita, > > One question that comes up immediately is why you are using DDM @ 5% > concentration. If it's a typo, understandable. I figure most of us use DDM > at 0.05-0.1% concentration, and I've used as low as 0.015%. > > Also, as others have pointed out, until you shoot it, you don't know. But if > it's hexaganol with very smooth/non-sharp edges, many of us have seen it > using DDM. > > -john > > > On May 4, 2012, at 7:48 AM, anita p wrote: > > > Hi All, > > I would like to have your expert advice on crystals. > > I am using detergents as 5% (w/v) of DDM 0.4ul in a 4ul (protein + > > condition + detergent). The precipitant is 28% peg 20K > > After 1 day I am able to see little plates of irregular shape . > > > > I am able to see some needles if I change into MEGA-10. > > > > I am able to see some hexagonal 3D crystals when I change the precipitant > > to Ammonium sulphate and the detergent to Anapoe-C10E6 > > > > Is there a possibility to check whether this crystals are protein or of > > detergent, even before shooting it ? > > Does the control drop test help ?? > > > > awaiting for feedback > > Anita > > > <IMG_8737.jpg>
