Dear Amala,
The first thing I would try is to run your protein sequence on this server: http://xtalpred.godziklab.org/XtalPred-cgi/xtal.pl. This is a webserver that will compare you aa sequence to other previously crystallized proteins. The result will also suggest which parts of your protein should be removed (if there are disordered regions). Have in mind that a bad score does NOT mean that your protein will not crystallize.
Protein crystallization is not the rule, it is the exception. You must be aware that some proteins will never give you crystals, no matter how much you try. And unfortunately, you can only know it after trying.
As people already said, always use fresh protein in your screenings and, if possible, purify it by SEC to ensure that you have homogeinity in your sample. If your protein is very soluble, I would even increase the protein concentratrion (in my group we have got better diffracting crystals using 25mg/mL)
Good luck
Rafael Marques da Silva
Mestrando em Física Biomolecular
Universidade de São Paulo
Bacharel em Ciências Biológicas
Universidade Federal de São Carlos
phone: +55 16 99766-0021
"A sorte acompanha uma mente bem treinada"
________________________________________________
De: amala mathimaran<mailto:[log in to unmask]>
Enviado:sexta-feira, 27 de dezembro de 2019 10:31
Para: [log in to unmask]<mailto:[log in to unmask]>
Assunto: [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
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