Hi all, I have a pure protein and i want crystallize it. My proteina is a high molecular weight (1.300kDa) and i want know the best protein concentration and conditions for crystallization this protein. Buffer: NaCl, Tris and Glycerol 10% pH:7,6
Thank you.
Hi David,
It sounds like this is your first time crystallizing a protein. In short, every protein is unique and there is no one concentration or crystallization condition that anyone can recommend. This is a very broad area, and so I have provided some links to some very good beginner's guides for crystallization. If you can afford a text book, I highly recommend Dr. Bernhard Rupp's "Biomolecular Crystallography" text.
I can tell you that you will have problems getting crystals due to your current protein buffer. Glycerol and higher concentrations of salt will generally prevent crystallization. You should try to store your protein in a buffer at the pH it is most stable at, and with as little salt as it will tolerate. Glycerol should be kept to a minimum and I recommend keeping the concentration below 1% (I typically used 0.1%). If you have already frozen your protein in the other buffer, you can use a dialysis button or a small desalting column to exchange the buffer to one that is better suited for crystallization. Sometimes, proteins can aggregate after defrost, so be sure to pellet any precipitation that may have accumulated during defrost and buffer exchange. It may be wiser to use size exclusion chromatography, as it will allow you to exchange the buffer, and remove any aggregation.
Good Luck!
http://www.ruppweb.org/Xray/101index.html
http://www.moleculardimensions.com/shopcontent.asp?type=tech_tips
http://www.proteinstructures.com/Experimental/Experimental/protein-crystallization.html
Hi David,
It sounds like this is your first time crystallizing a protein. In short, every protein is unique and there is no one concentration or crystallization condition that anyone can recommend. This is a very broad area, and so I have provided some links to some very good beginner's guides for crystallization. If you can afford a text book, I highly recommend Dr. Bernhard Rupp's "Biomolecular Crystallography" text.
I can tell you that you will have problems getting crystals due to your current protein buffer. Glycerol and higher concentrations of salt will generally prevent crystallization. You should try to store your protein in a buffer at the pH it is most stable at, and with as little salt as it will tolerate. Glycerol should be kept to a minimum and I recommend keeping the concentration below 1% (I typically used 0.1%). If you have already frozen your protein in the other buffer, you can use a dialysis button or a small desalting column to exchange the buffer to one that is better suited for crystallization. Sometimes, proteins can aggregate after defrost, so be sure to pellet any precipitation that may have accumulated during defrost and buffer exchange. It may be wiser to use size exclusion chromatography, as it will allow you to exchange the buffer, and remove any aggregation.
Good Luck!
http://www.ruppweb.org/Xray/101index.html
http://www.moleculardimensions.com/shopcontent.asp?type=tech_tips
http://www.proteinstructures.com/Experimental/Experimental/protein-crystallization.html
Without any further information the only advise I can suggest is you go for batch crystal trials and screen for hundreds of variations in the buffer and salt strengths and composition until you get crystals. It seems tedious and disheartening but don't worry now with the advent of robots in crystal trials, things are not as scary (apparently) as they were fifty years ago.
Hi David,
Elizabeth gave you some good advice regarding your buffer composition but I am slightly worried about the size of your protein. You mention your protein is 1.300 kDa in size and this is either very small or simply enormous.
I know that punctuation in numbers is used differently in Spanish and in English speaking countries and while the decimal indicator in English is a point and the visual aid to separate large numbers into thousands, millions, etc... is a comma, in Spanish it's the other way round (e.g 1.5 in English is written as 1,5 in Spanish and 1,300,000 in English is written as 1.500.000 in Spanish). Therefore, I am wondering if you mean 1.3 kDa (about 10-12 amino acids) or 1300 kDa (about 12400 amino acids).
If your protein is over 12000 amino acids in length, it's going to be a large multidomain protein with one repeating or many completely different domains. Multidomain proteins are notoriously difficult to crystallise as there generally is a degree of flexibility between the domains, which is not good for forming the rigid structures that are needed to produce diffraction-quality crystals.
Don't worry too much about this, though, as this is not impossible (some viral capsid structures are HUGE and they've been solved by crystallography), so you should definitely give it a try. However, if this doesn't work it's better to try and crystallise the individual domains of your protein separately. You can then try single particle or cryo EM or even SAXS to assemble the single domains into a full-length structure.
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