Re: [ccp4bb] Concentrating a protein solution - subbu
A solution could be to control the solubility of your protein in different pH and salts (you can also add some additives) by using the Thermal Shift Assay. You may find a better buffer in which to concentrate your protein. If you think that the reason of the low diffraction is the quality of your crystals try the suggestion of Eric, but if you think that is due to the small size of the crystals, you can also try to make drops of 3µl (2µl of protein and 1µl of the well) or to feed your crystals during the growing (i.e. adding 0.5µl of protein solution to your drop after when you see that the crystals stop to grow), or to do seeding like Eric suggests, but in big drops (5µl in hanging or more in sitting drop) and with serial dilutions of the seeds. Good luck... Michel. Dear All: We have been trying to crystallize a protein which is large - 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that others use to increase the concentration other that using amicon columns. Any help will be appreciated. Thanks Subbu
Re: [ccp4bb] Concentrating a protein solution - subbu
Subbu, Don't forget you crystallization screen can also be used to find a condition that your protein may be happier in ! As you already have setup a screen of your protein, have a look at your drops again and see if there are any conditions that are clear. If so, is there a consistent theme in the conditions i.e pH or additive ? I've worked with one protein that behaved similarly and we saw that in drops at pH 5 or below it remained clear. Upon changing the purification procedure to this lower pH we could concentrate the protein up to 40 mg/ml (and got nice crystals) instead of the borderline 2mg/ml we were getting at pH 7.4 Goodluck Stephen On 21 July 2011 17:53, Narayanan Ramasubbu ramas...@umdnj.edu wrote: Dear All: We have been trying to crystallize a protein which is large - 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that others use to increase the concentration other that using amicon columns. Any help will be appreciated. Thanks Subbu
[ccp4bb] Concentrating a protein solution - subbu
Dear All: We have been trying to crystallize a protein which is large - 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that others use to increase the concentration other that using amicon columns. Any help will be appreciated. Thanks Subbu
Re: [ccp4bb] Concentrating a protein solution - subbu
You could try loading a small 1ml HiTrap (or similar) Q or S column with your protein - and knocking it off it in one go with high salt, alternatively micro-dialysis against a solution containing PEGs can also work well. Tony Sent from my iPhone On 21 Jul 2011, at 17:54, Narayanan Ramasubbu ramas...@umdnj.edu wrote: Dear All: We have been trying to crystallize a protein which is large - 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that others use to increase the concentration other that using amicon columns. Any help will be appreciated. Thanks Subbu
Re: [ccp4bb] Concentrating a protein solution - subbu
Hi Subbu, You got crystals at 1mg/ml so you probably don't need to concentrate your protein any higher, especially since you suggest that concentrating beyond that is problematic. Instead, you may want to try to optimize the crystallization condition you have already identified. Some possible things to try: additive screen, include specific ligands, different temperature, different ratios of protein solution to crystallization solution, seeding, different crystallization methods (hanging vs. sitting drop, batch, diffusion,...), ... good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Thu, 21 Jul 2011, Narayanan Ramasubbu wrote: Dear All: We have been trying to crystallize a protein which is large - 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that others use to increase the concentration other that using amicon columns. Any help will be appreciated. Thanks Subbu
Re: [ccp4bb] Concentrating a protein solution - subbu
See below. I believe Ray intended this to be sent to the entire bb and especially to Subbu! And in reading Ray's message, I am reminded that I forgot to mention what he may be hinting at. You should also try to optimize by creating a fine grid screen around your identified condition varying in particular pH and precipitant concentration. best, Eric On Thu, 21 Jul 2011, ray-br...@att.net wrote: What is the pI? Perhaps the protein will be more soluble close to this pH? You could increase the salt and/or add some glycerol or a cofactor? Normally crystallization is most affected by the pH, 2 - 10% glycerol or temperature 4 - 25 degrees C. Cheers Ray Brown _ From: Eric Larson larso...@u.washington.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Thu, July 21, 2011 1:40:51 PM Subject: Re: [ccp4bb] Concentrating a protein solution - subbu Hi Subbu, You got crystals at 1mg/ml so you probably don't need to concentrate your protein any higher, especially since you suggest that concentrating beyond that is problematic. Instead, you may want to try to optimize the crystallization condition you have already identified. Some possible things to try: additive screen, include specific ligands, different temperature, different ratios of protein solution to crystallization solution, seeding, different crystallization methods (hanging vs. sitting drop, batch, diffusion,...), ... good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Thu, 21 Jul 2011, Narayanan Ramasubbu wrote: Dear All: We have been trying to crystallize a protein which is large - 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that others use to increase the concentration other that using amicon columns. Any help will be appreciated. Thanks Subbu
