Dear Amala, You have already received a lot of good advices from expert crystallographers. Apart from the notables, i would still read about the protein. Is this an enzyme? If yes, what is the ligand or a co-factor? The protein conformation would be different in apo form versus bound form. Often, the protein adopts a more stable/rigid conformation upon binding to its ligand/co-factor, and this addition of ligand/co-factor eventually helps in crystallization (a floppy region in a protein is recalcitrant towards crystallization, hence a ligand will do!). Likewise, is the tag cleaved? Also, is there any free cysteine present in your protein that may engage in a disulfide bond? You may want to add a reducing agent to protect the cysteine. Alternatively, one may harness the power of (non-physiological) disulfide bridge to stabilize the protein of interest. For reference, you may read my manuscript, Khan et al, (2014) J. Virol. ( - DOI: - 10.1128/JVI.03455-13 <https://www.researchgate.net/deref/http%3A%2F%2Fdx.doi.org%2F10.1128%2FJVI.03455-13?_sg%5B0%5D=48r7xs6TPbET5n5PSde4APDbG-dQM6CPVkGr6OvkXkEjoGTfbv_xjLNQVNG2smilwfagzSutIjn_J1Kgz_sGJWtoLw.XpTr2Kuw_FgXXPENkyPdqnROiZfMuot08FeXIH6M84pZsKBdqky8zXeSsRuU3qGeW31m0S5iudcKJPDKMp2Qrw> )
Further, i would invest a little time in database search. You may aim to find the structure(s) of the homologous protein(s) in the protein data bank. This will allow you to gain confidence in the (1) domain boundaries of the expression cassette; (2) buffer composition of the final protein prep; (3) selection of the ligand/co-factor; final protein concentration; and (4) crystallization condition (homologous proteins often tend to crystallize in the similar range of the pH and precipitant). Eventually, you may procure a more focused crystallization screen (such as JBS kinase screen for kinases). happy voyages! z Best wishes -Z Zaigham Khan, PhD Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York On Fri, Dec 27, 2019 at 8:31 AM amala mathimaran <[email protected]> wrote: > 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
