Hi! I had a similar problem with an overexpressed protein a couple of years ago, however mine was still detectable in the insoluble fraction.

I'm assuming this is a recombinant protein, have you confirmed that your protein of interest is actually being expressed?

I used this method outlined in this article to solubilize and purify my protein and had great results. Hope this helps

Article Use of anionic denaturing detergents to purify insoluble pro...

Did you try 6-8 M urea in your buffer instead guanidine? Also, it is important to check the pI of your protein and confirm that you can use pH 8 in your buffer. Good luck.

Hi Kiyohiro,

could you give some more description of your protein, the nature of the construct, tag if used, hydrophobicity and pI (as suggested by Maxuel above) and the cell strain you are using?

Secondly, at the moment, can you confirm that you are purely looking at cell lysates by SDS-PAGE to show that the protein is mobilised and where it has gone rather than trying to capture a protein on some form of solid substrate ie a resin? As Zainab has asked above, do you have a gel showing the induction of the protein and stages of extraction - proteins don't just disappear, so if it is in the cells to begin with, it must have gone somewhere in the processes above.

The reason I am asking about hydrophobicity stems from one tagged protein I was purifying a few years ago where a hitherto unexpected characteristic I found was that when cleaved off the tag (which purified it beautifully to that point), the protein disappeared instead of flowing off the column - we could subsequently elute the tag cleanly. After a lot of SDS-PAGE taking samples from every part of the process, I eventually realised that it was sticking near irreversibly to the material from which the column was made due to a domain of very high hydrophobicity.

Happy to keep discussing this. Best of luck, Robin