The diffraction you see is probably not the best diffraction you could obtain from these crystals. I have found long thin needles are very susceptible to manipulation. I would highly recommend seeding (I like the Hampton Seed Bead personally, http://hamptonresearch.com/product_detail.aspx?cid=18&sid=44&pid=42 ). You need to try to change the shape of these crystals to beef them up.
Other Recommendations: Vary protein concentration Vary pH (if there is not a buffer in the well, determine pH of well and add buffer to stabilize pH and screen around - I've had success changing needle clusters with a few 0.1 pH unit changes) Dioxane to limit crystal nucleation, try to only add it to the well, not mother liquor present in drop, 1 - 5 % Dioxane Different MW PEGs There are almost limitless possibilities of things to try with protein crystallography. Good luck, Kelly Daughtry ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Tue, Jul 24, 2012 at 9:40 AM, Niks <nik...@gmail.com> wrote: > Dear All, > > I am trying to crystallize a recombinant dehydrogenase protein. Got five > hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M Sodium > Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M sodium formate > and 0.2M Potassium Chloride) after two days. > Crystals looks like needles most of time, sometime broader needles > (Pictures attached). UV crystal scanner says those are protein crystals, > but when we tried to pick up one and shoot at room temperature, diffraction > patterns looks like similar like of powder diffraction (picture attached). > I have tried 50 of the 96 additives(whichever I can arrange of) mentioned > in the additive screen from Hamptons . I have tried detergent screen from > Hamptons (this time original screen solutions). I have tried incubating the > plate at 28degrees as well as 10degrees, Though waiting for 10degree > results but one drop showed needles again after normal two days of growth > period. > I tried to slow down the supersaturation by adding 100ul of 1:1 ratio of > silicon oil and paraffin oil over the 1ml of well solution. This time no > crystals but some precipitation. > > If anyone spare any word of wisdom to improve these crystal quality, I > will be very grateful. > If seeding is the only obvious thing to try, any reference for the seeding > procedure will be highly appreciated. > > Thanks very much > Nishant Varshney > PhD student, > National Chemical Laboratory,Pune,India > -- > "The most difficult phase of life is not when No one understands you;It > is when you don't understand yourself" >
