I'd suggest you play around with the TX-100 and CHAPS concentration but a big factor is time. When I before started to refold the protein with TX-100 and CHAPS and bound it straight to glutathione sepharose beads (batch binding) for 1-2h, I hardly recovered any protein. Allowing the refolding/binding to beads to happen overnight (4C end over end rocking) increased the yield significantly (recovered ~90% of the protein). Good luck!

Many protocols I've seen use detergent to wash their pelleted inclusion bodies of contaminating cell junk (followed by a wash with no detergent) and then incubate them with 8M urea or 6M guanidine solutions (maybe 60 min at room temp for starters). I've puttered with a few inclusion body constructs and like the urea route better because you can do SDS-PAGE directly from aliquots from various stages of the process (make it fresh for every try). From there it seems like your next step would be dialysis. People have had success with the "infinite dilution" method, slowing adding your unfolded supernatant to a large volume of mixing buffer drop by drop, or simply slapping your dialysis bag into buffer like normal. If both of those aren't working very well for folding you could try moving your dialysis bag to buffers with increasingly lower concentrations of urea (maybe 4M, 2M, 0M etc).

Lastly, His-SUMO fusions seem to be a good choice for many.

I would be nervous about keeping my proteins in buffers at their pI. A little charge and go a long for solubility.

Does anyone have some experience with His-MBP tag? will it help?

simply, you should try equilibrating the HIS- beads by using the same buffer with the same amount of detergents, for example during 1-2h, before to incubate with the protein extracts for binding?. Similarly, the washing buffers and elution buffer must be supplemented with the same detergents.

Hi, I used 0.1% sarkosyl in my lysis buffer and I bound perfectly GST fusion proteins O/N at 4°C by putting the pre-washed gluthathione beads in the same extraction buffer without sarkosyl. I also add 0.5% Triton X100 it do not affect binding. However I put 1mM final PMSF and cocktail inhibitors.

Hey rafael I have used His-MBP tag and it certainly increase the protein solubility at some extent but of course it may vary protein to protein. another thing i feel that possibly you have used too much sarcosyl which have denatured your protein. try to use 0.1-0.2 % of sarcosyl it should not affect the GST binding. Good luck

We have used successive steps of a Nickel column followed by ion exchange to purify a sarkosyl-solubilized protein. Sarkosyl is charged so you need to use the ion exchage column with the same charge. I don't think you will remove the sarkosyl completely with one chromatography step unless you wash with a buffer like CHAPS while it is on the column.

Try to replace your protein solution with detergent to the binding condition of the chromatography beads with a gradient carefully. If possible, static adsorption experiments before chromatographic experiments are useful for optimizing adsorption and elution conditions. PS: pH and salt concentration are important.

i used to solublize the membrane protein with sarkosyl. on purification the protien is not binding to GST beads may be due to unfolding. so what could be best possible way to avoid this problem

I'd suggest you play around with the TX-100 and CHAPS concentration but a big factor is time. When I before started to refold the protein with TX-100 and CHAPS and bound it straight to glutathione sepharose beads (batch binding) for 1-2h, I hardly recovered any protein. Allowing the refolding/binding to beads to happen overnight (4C end over end rocking) increased the yield significantly (recovered ~90% of the protein). Good luck!

I have been using the same paper. I find the best combination was solubilized the sample in 1% sarkosyl, then add 2% triton x100 and 20mM Chaps

I can give you more details if you need too.

In addition, look the papers mentioned below

Improved recovery of active GST-fusion protein...

Solubilization and purification of enzymatically...

One more thing; I found that sonicating my sample in the presence of sarkosyl "ruined" the protein, it didn't refold and didn't end up binding to the glutathione beads. So, add sarkosyl after sonication and it should significantly increase your yields.

After protein solubilization by sarkosyl, it should be removed in case it will impact GST fusion protein binding to the sepharose. Combination of Triton and CHAPS can effectively remove sarkosyl from GST fusion protein which permits protein refolding and binding to the sepharose. It is better to incubate the GST fusion protein with beads for more than 6 hours to improve yield of protein. I have solved this problem I met with application of Triton and CHAPS.