2 replies to this topic

[braindrayn]

Hi everyone! I've been having trouble purifying a protein on a cation exchange column and have turned to the internets for insight. My protein has a calculated pI of 8.9 and I've tried running it on a cation exchange column using a sodium acetate buffer system. It stick to the column fine because there is no flow through but nothing elutes off the column when I start the salt gradient! It in fact takes a solution of 8M Urea to knock my protein off the column when I need to regenerate it. I'm getting my protein from a solid phase synthesis system so even when I am well below the pI I am unable to solubilize the entire powder probably due to a mix of other proteins present in the system. Any one have any tips or tricks on what else I could try to possibly purify this protein?

Thanks.

[protolder]

[quote name='braindrayn' timestamp='1307827977' post='112362']
Hi everyone! I've been having trouble purifying a protein on a cation exchange column and have turned to the internets for insight. My protein has a calculated pI of 8.9 and I've tried running it on a cation exchange column using a sodium acetate buffer system. It stick to the column fine because there is no flow through but nothing elutes off the column when I start the salt gradient! It in fact takes a solution of 8M Urea to knock my protein off the column when I need to regenerate it. I'm getting my protein from a solid phase synthesis system so even when I am well below the pI I am unable to solubilize the entire powder probably due to a mix of other proteins present in the system. Any one have any tips or tricks on what else I could try to possibly purify this protein?

Thanks.
Hola, It is very important in all types of cromatographies have the sample clear and if it´s possible filtered throught 0.22/045um to be sure that your protein is soluble. if you can´t filter, a high speed centrifugation gives you a clair extract for column.you have your protein precipitated inside the column and for that it´s eluted with urea, so it was precipitated from the begining or any change in the pH leads to precipitation.so check that your sample is in the same buffer conditions that colum and check its presence after filtering or high speed centrifugation (50.000g 30min). if no first check the solubility probability of it http://www.biotech.ou.edu/or http://mips.helmholtz-muenchen.de/proso/proso.seam and try to solubilize and purify with urea/reducer followed by refolding, if yes try to use a not very acid pH as 6 with phosphate.  Buena suerte

[kidroc]

If you run a AKTA system to purify, you may also notice a system pressure increase when you sample starts precipitating inside the column. I will also suggest you filter or by other means to clean you sample before loading to the column. Good luck.