Hi Sajid, We need a bit more info to help you.
1) What sort of columns/techniques did you use? Affinity chromatography (nickel, GST, MBP, etc...), ion exchange (cation, anion ... heparin basically falls into this category as well), size exclusion, etc ...? 2) What were the exact compositions of the buffers? 3) Conditions of the runs (temperature, time between lysing the cells, first column and second colum, etc...) 4) What sort of protein are you trying to purify (give us a general type of enzyme/protein class, you don't need to be specific if you don't want to)? Many types of protein have certain needs/preferences that you might not know about but somebody on the bulletin board might point out for you. Finally, you should alway run "load", "flow through" and "elutate" samples from your column runs on SDS-PAGE. Did you do this and, if so, was your protein of interest present in the "load" sample (the pooled flow through sample you obtained from your first column)? It might have degraded or maybe it stuck to the filter/concentrator membrane if you filtered/concentrated the sample between columns. Regards, Tony ------------------------------------------------------ Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE ________________________________________ From: CCP4 bulletin board [[email protected]] on behalf of sajid akthar [[email protected]] Sent: 18 January 2017 11:32 To: [email protected] Subject: [ccp4bb] Protein Purification Dear All I am purifying a protein from liver. In first step I spotted protein in Flow Through and could see intense band in SDS PAGE. I pooled the fraction and did second column. Surprisingly I could not see UV absorbance or even any band in the SDS. I used PMSF as protease inhibitor from begining of purification. First column I used Tris buffer (pH 8) and for second column I used MOPS buffer (pH 6). What could be the reason? Thanks in advance Sajid
