Well, there is no a priori answer what will help in your case. Most probably, you will have to try all:-) In general, pH may have the largest effect as you change the electrostatics. Also you may try the additive screens, to see if they help. Furthermore, depends on how many screens you've tried already - if only 1-2, try more, if already 5-10 and you see only this, that probably means your protein is unhappy. You may need to use another construct (chopping the termini, or loops, or his-tags, etc) or in a worst case you should start looking at orthologs - quite often the same protein but from another organism may crystallize easier. So lot's of things to try - good luck!

When I was trying to crystallize  EmrE (in 0.02% DDM, membrane protein)

I was getting a lot of spherulites. Addition of amphiphilic additives

(heptantriol, i-BuOH  etc 0.05-0.5%) helped to obtain diffracting crystals.

Never got something good enough to solve the structure though :( 

Zaiddodine,

There is an a priori answer that will help you - if anything can.  Set up "random" microseeding, i.e. make a seed stock and add it to a couple of random screens.  If anything in those wells is crystalline or ordered it's likely that:

(1) You will pick up new crystal hits in the screens

(2) The crystals will be more ordered than in normal screens

(3) You will be able to control the number of hits per well by diluting the seed stock

The SPH wells look hopeful to me.

It's very strange that this method is taking so long to catch on!

Best wishes

Patrick

________________________

See 

[1] D'Arcy, Allan, Frederic Villard, and May Marsh. "An automated

microseed matrix-screening method for protein crystallization." Acta

Crystallographica Section D: Biological Crystallography 63.4

(2007): 550-554.

[2] Further information on the theory and practice of the MMS

method is available at the Douglas Instruments web-site,

http://www.douglas.co.uk/mms.htm and http://www.douglas.co.uk/MMS_proc.htm

[3] Shaw Stewart, Patrick D., et al. "Random microseeding: a

theoretical and practical exploration of seed stability and seeding

techniques for successful protein crystallization." Crystal Growth &

Design 11.8 (2011): 3432-3441.

[4] Abuhammad, A., Lowe, E. D., McDonough, M. A., Shaw Stewart,

P. D., Kolek, S. A., Sim, E., & Garman, E. F. (2013). Structure of

arylamine N-acetyltransferase from Mycobacterium tuberculosis

determined by cross-seeding with the homologous protein from

M. marinum: triumph over adversity.Acta Crystallographica Section D:

Biological Crystallography, 69(8), 1433-1446.

[5] Obmolova, Galina, et al. "Protein crystallization with microseed matrix

screening: application to human germline antibody Fabs." Structural

Biology and Crystallization Communications 70.8 (2014).

Patrick, I will completely agree with you, if it is a soluble protein. For membrane proteins MMS is still something non-trivial. But of course we don't know what kind of target Zaidddone has, so I hope he has enough information now to think about. 

I was in your shoes not too long ago, obtaining spherulites. If my understanding of what you said wasn't wrong, I recommend using a few more screens. 50 buffers isn't a lot, since each screen set has 96. I typically go for 5 sets of screens. In addition, do see if hits have any common components between them, e.g., common buffer or pH. This gives a clue on which screen to use next. 

But if you're feeling particularly stubborn (it's not a bad thing), I agree with Albert on using Additive Screen - it usually helps. In addition, it may be a good idea to use a substrate as well, since it can stabilize the protein and might even give rise to a better crystal form. You're getting there soon, keep at it! :D

Misha, that pain, we all know too well. Hang in there. 

I had a similar problem earlier this year and after some literature searches found a comment saying that gelatinous precipitate seems to be more common in drops set up at temperatures above 18oC using protein samples stored in buffers with high ionic strength >200 mM NaCl. I think it was on the Hampton website where I read this ... but don't quote me on this. Anyway, they mentioned that the amount of gelatinous precipitate can be reduced by lowering the crystallisation temperature to 4oC and reducing the salt concentration of the protein buffer solution. My problem was that my full-length protein needs at least 250 - 300 mM NaCl to stay in solution and I eventually side stepped the problem by truncating it. This resulted in a more stable product and allowed me to store my protein in buffer with 100 mM NaCl, after which I got no gelatinous precipitate at all.

I'd be interested in finding out if reducing the NaCl concentration works for other people (as in my case it could have been the truncation that affected the gel formation rather than the lower NaCl concentration). Give it a try and let us know what happens.