Hi all,
I am having issues with protein precipitation during buffer exchange. His-tag protein is eluted in 20mM HEPES, 200mM imidazole, 500mM NaCl at pH8.0. During elution, the tube with the highest amount of protein >80uM was cloudy and the remaining tubes with 60uM (1.8mg/ml) were fine! I do not want to concentrate the protein any further as the amount is high enough for my assays. During buffer exchange (20mM HEPES, 100mM salt, pH 8.0) protein started to precipitate. I am wondering if the precipitation is caused by desalting?
What is the pI of your protein? How did you perform buffer exchamge?
Some proteins require high salt to prevent precipitation when they are concentrated. A change in pH can be helpful, as well as maintaining the high salt condition in the concentrated stock solution. When the protein is diluted, the high salt may no longer be necessary.
Hi Omar and Adam,
Thanks for your replies. Theoretical pI is 6.8 without the His-tag. I used vivaspin 6 concentrator to perform the buffer exchange. I wasn't concentrating the protein. I just wanted to desalt the protein! I used 500ul of 50uM protein and topped up with the new buffer to 4ml to lower the salt (100mM). The tubes were spun down to 1ml (so I should now have 25uM of the protein). I repeated this again with the low salt buffer and protein started to precipitate.
As Adam says it is a concentration problem then, follow his recommendations
It may be problem of the His-tag. Check the paper by Zhu et al 2012
https://www.ncbi.nlm.nih.gov/pubmed/23250226
Hi Kiran, don't you think you were concentrating the protein when you were spinning down the 4 mL (100mM salt) sample to 1mL ?
@Tomas, thank you! I will have a look!
@Chen, thank you! I am not concentrating the protein. To start with, I had 50uM of 500ul protein. I topped up with my new buffer (100mM NaCl) to 4 ml and spun down to 1 ml. In fact, I diluted the protein! Theoretically, I should have 25uM (1:2 dilution)!
It is often like this because imidazole stabilizes proteins as arginine and glutamate do so keep some inside or add Glycerol in time e.g. in the exchange buffer. This can especially happen if you change also the NaCl which might be important as well.
you are adding low salt buffer directly to the protein in high salt buffer. so there is slat concentration shock to the protein. better you use dialysis tube to slowly dialyze out the salt. this may work. good luck
Your protein could be binding some stabilising co-factor like an ion or small molecule and losing it as you do the buffer exchange.
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