Dear Wei,

I have many experience for insoluble recombinant RNA-binding proteins, although not from insect cells but from E. coli.  But it may be applicable for insect cells either.

My successful case was first fully denature insoluble protein in 6 M urea and purify it under buffer including 6 M urea by column chromatography (for instance Mono Q and Mono S).  Once it was purified then many contaminants, such as RNA and lipid, are removed then the solubility of the protein becomes much better.

In the final step, your purified recombinant protein should be dialyze gradually with buffer including 25% glycerol, which is also the trick.

I hope this information helps for your problem.  If you have further questions, please ask me anytime through my e-mail address

Best of luck,

Akila

If your protein does not dissolve in 6 M guanidine HCl or guanidine isothiocyanate, then I don't think there is much hope for getting it in soluble form. If there are non-membrane domains that you could express separately from the rest, you might have better luck getting something soluble to work on.

How do you plan to do mass spectrometry on an insoluble material?

Dear Akila

Thanks so much for your valuable information. I will try what you suggest. Otherwise, I may bother you by sending you an email.

Thanks

Wei

Dear Adam

That is also what we are going to propose by expressing fragment of the protein. with insoluble protein, I am struggling to do MS. Do you have any suggestions?

Thanks

Wei

You may be able to dissolve the protein in hot SDS-PAGE sample buffer. Then you can run it on a gel and cut out the band. The protein can be digested with trypsin, and mass spec can be used for identification and sequencing.

Hi Adam

we are trying to use top-dowm approach to do MS. trypsin digestion may not work for this approach. but we may also do bottom-up MS with trypsin digestion.