Purification of gst and His tagged protein - after purification there are still many contaminated bands (Oct/30/2008 )

Dear you,

I have many problems with purification of my proteins. I want to purify my modified protein from the unmodified proteins. The modified proteins has His tag as well as GST tag, while the unmodified protein has only GST tag:

The first problem: I have tried using 4B beads to pull down gst tagged proteins (including modified and unmodified ones). At this step I found that 4B beads did not work well because when I checked the protein purity by SDS-PAGE there were still many many bands beside my desired proteins. I used PBS1X plus 0.75mg/ml lysozyme as the lysis buffer. Add 1mM PMSF before sonication. Then I washed the beads (column) with 10-20 volumes PBS1X for total 3 to 4 times. Finally, I eluted by 10mMGSH in 50mM Tris pH 8.0. Therefore, I also tried with washing by 10mM Tris, 1mMDTT, 0.2M NaCl, pH 8.0 and eluting by 10mM GSH in washing buffer + 1mMEDTA , pH 8.8. However, it did not help to improve the purity of proteins.

The second problem: I try to combine some techniques to purify the modified proteins: After using 4B sepharose beads to pull down all gst-tagged proteins (including modified and unmodified ones), I used Hitrap Q-column to get purered proteins and then used Nickel column to pull down the modified proteins. However after doing Western Blot there is still the unmodified protein there beside the modified protein.

Then I tried to combine 3 methods: firsly 4B beads, secondly Nickel column, and finally Gel giltration (using Superose 12). Unfortunately I still cannot get rid of the unmodified protein from modified one.

Could any one give me some suggestions to improve the purity of my proteins? hank you so much!

you could try hydrophobic interaction chromatography, chromatofocusing,...

see what ideas you can get from the protein purification handbook (ge healthcare).