Hello Meg, Since your protein is quite acidic, your next step could be e.g. anion exchange - provided that you are able to get the protein into a suitable buffer w/o losing it (since you will pass through the pI). If you can, the simplest way to do so is to add TRIS to the pH4.5 buffer until you get the desired pH (7-8) and dilute the sample a bit with pure water.
An alternative, or a follow-up step could be hydrophobic column - these are very useful for removing aggregates, partially folded stuff and similar entities. Again, nothing is completely safe - HIC may cause problems since you are starting at fairly high salt (1-2 M NaCl or KCl typically). But at least you won't have to pass the pI. Cheers, Artem > 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. > >
