Dear Amala,
There are some good suggestions coming your way from here. I would also recommend checking out the High-Throughput Crystallization Screening Center at the Hauptman-Woodward Medical Research Institute or other similar services. They run most of the common types of screens in an efficient and cost effective manner. There is good advice available on how to interpret the outcomes.? Our Center (http://getacrystal.org) has a section on crystallization research https://hwi.buffalo.edu/high-throughput-crystallization-center/crystallization-research/ and there are links to papers that describe how to make some sense of results from initial crystallization screening and how to optimize those results. I'm biased but the advice is good and the references many of those papers contain are comprehensive. I've been involved many of these studies and have copies of some of the full papers on my own website at https://hwi.buffalo.edu/scientist-directory/snell/ - just click on the Crystallization Methods and Analysis section. I would recommend looking into the solubility phase diagram for crystallization. That would give you an idea of the influence of changing different parameters. You do have some common outcomes in the results you showed which define a pH range, salt, and exclusion agent concentrations. Also small changes in pH can have a dramatic outcome. Increasing the protein concentration may push you further into the nucleation region and may result in far more but smaller crystals - which could be mistaken for precipitate. Screen around each condition and try to understand the impact of each axis and the overall combination of those axes. I think the results you showed bode well and it shouldn't take to much work to optimize those conditions. I would also take a UV image to confirm protein or spin some of the crystals down in a capillary and take a powder pattern to confirm protein crystals and not the ligand or crystallization screen component. Best of luck, Eddie ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of amala mathimaran <amalat...@gmail.com> Sent: Friday, December 27, 2019 8:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to get protein crystal Dear all, Can you suggest me how to get protein crystal??? I purified my target protein and concentrated to 12mg/ml (pI 6.04). Final purified protein contains 50mM HEPES pH 8.0 and 50mM NaCl. Then the initial screening was done using hanging drop method but no crystal. So 2mM NADP cofactor was added again screen with Hampton (PEG ion, PEG RX, crystal screen, Index) and Molecular dimensions conditions etc. I got precipitate like formation the image was attached below. From this formation what I can do... mean while I increase the protein concentration and did screening for that selected conditions again I got same kind of formations. I am new to protein crystallography kindly suggesting me. And how much concentration is suitable for protein crystallization?? How to find which concentration is enough for our target protein crystallization?? Thanks in advance ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
