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.

I used to use Tallon, which worked good for me

When you purify using a IEX colunm you select the sample (the peak) by the surface charge. All proteins that have a similar Isoelectric point as your protein will be co-purified. Obviously if you start from inclusion bodies most of the other E. coli cytoplasmatic proteins are not a problem but some of then can by purified with them (with the inclusion bodies). I think you have two problems or solutins.
1) You are staining with silver stain than can reveal band of proteins that are really in small quantity. Usually with this stain, principal bands saturated fast, and the bands in lower concentration appear. Its makes looks as you have more contamination than really you have. I think you make a gel again loading at leas 20 or 50 times less protein to see the real proportion of the contaminant in the sample, and if this is too high for your purpose. Its looks less than 2% of contamination. Do you need a protein more than 98% pure?
2) You need a more pure protein:
a. If you have a protein expressed with a tag, like GST, His, etc use a affinity resin
b. If not, you can use to a resin for molecular exclusion chromatography
Good luck

hi,

As u said you have done ib ,refolding, IEX did u play with washing the IB with some buffer if yes, did u try with detergents some times it can also do u better job.

.....And you can try hydrophobic chromatography.

....since your protein also looks >90 % pure u can try with superdex 75 if your protein size is ~30kda , 200 if more than 35kda.