My target protein is extracted in PBS, is eluted from column in DAP and neutralised with Tris-HCl, then dialysed against PBS resulting in 4mg/ml. However as I want to do an initial crystal screen I used a pre washed centricon to exchange buffer to Tris and subsequently have a much reduced concentration (1.4 mg/ml) and have lost activity. Any suggestions please? pI is 4.5, Tris & PBS are @ 7.2 pH, could it be to do with the molarity of the tris (10 mM)?
The possible reasons I can think of for low activity are:
the protein needs to form dimer, tetramer etc in order to have full activity
the protein died during the course of the concentration or you lost some cofactor upon concentration and dilution with tris.
your protein just likes PBS. It could be the higher salt. But I have seen where some proteins just like one buffer and not another for no apparent reason even though there is one.
you can do buffer exchange with desalting spin columns which are fast and easy. That way you could try multiple buffers to find the best one and keep the protein fairly concentrated.
If you start out with 10 mM tris as your buffer, you can add back components to find out what your protein needs for the best activity. This is assuming you don't permanently reduce the activity by what you do.
Hi,
your could run a thermal shift assay and see if your target protein is "happy" in the new buffer befor forcing it into the new environment.
Bufer exchange in a concentrator could lower the solubility border (due to the new buffer) and concentrating the protein at the same time. That crushes it out.
I would suggest testing the new buffer in thermal shift or DLS.
Good luck,
cheers
I agree with both of the above comments. Thermal shift is particularly helpful for crystallisation - sometimes the optimal buffer suggested by thermal shift can make a huge difference to the crystallisability of the protein (usually positively!). It sounds like your sample is mostly very good, so doing a little work to support it will give a good result.
The usual reference for thermal shift can be found at http://wolfson.huji.ac.il/purification/PDF/Literature/Niesen2007.pdf.
Good luck!
Thank you for your help Marcia, Sven and Nicholas. I have not used Thermal shift before but will investigate how to do that. Can you let me know Sven what you mean by DLS? whilst i research these techniques i will try to work with my remaining sample to see if i can recover activity. It sounds like maybe centricon is not such a good tool for buffer exchange? I use it for removal of low MW compounds instead of dialysis at the moment purely to save time and assumed it would be safe for exchange. Thank you again!
DLS is dynamic light scattering - a very helpful method for discovering whether your sample is mono-disperse (i.e. all the same apparent oligomeric state), or whether it has any aggregation. As I am sure you can imagine, aggregation will make crystallisation very unlikely.
Using centricons as you did ("diafiltration") can work, but the protein tends to be very concentrated at the bottom of the centricon (you usually have a gradient of concentration across your sample). Another quick method is to use buffer exchange columns (e.g. http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/17085101) - these don't work for all proteins, but give a complete buffer removal, and you really don't want any phosphate in crystallisation samples!
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