Another option is to purify in denatured condition, then refold. --Chun -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Meg Sent: Tuesday, September 23, 2008 12:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Troubleshooting protein purification cation IEX
Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient - 1 C.V., 35 -80% gradient - 10 C.V. and 60 -100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 -6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated.
