In order to find out if your protein is expressed in an insoluble form, you break open the cells (such as by sonication) and centrifuge the extract. Run portions of the supernatant and the pellet on a SDS-PAGE gel. If the protein is in the pellet, then it was expressed in an insoluble form. If you wash the dirty pellet in detergent and centrifuge it again, and the new pellet is white and your protein is in it, the protein is in the form of inclusion bodies. The inclusion bodies may be nearly pure overexpressed protein. The inclusion bodies can be dissolved in urea or GuHCl. The dissolved IBs can be run on SDS-PAGE, but care must be taken. The bands will try to spread sideways because of the high concentration of salt. Make sure the adjacent wells have the same amount of solubilizing agent as the sample to keep the bands from spreading.

If the protein isn't a membrane protein then I think that it should be conceivable to solubilize it with some trouble-shooting. One way to solubilize a protein is to add a tag like a Sumo tag. That can be done with PCR based cloning where Taq polymerase mischievously adds 3' A nucleotides, and there is a kit which contains a linearized pETSumo plasmid with T overhangs, and the free hanging "A" and "T" base pairs pair up. They say it is simple ... but 1-pair of nucelotides pairing up is not that entropically favorable. After protein expression and purification, the Sumo tag can be cleaved with Sumo protease to get your free protein, and Sumo and Sumo protease have His tags which bind to the final nickel-NTA resin and your protein is collected in the flow-thru.

There is also ways to optimize protein expression like less IPTG induction concentration is more. (I would suggest

Inclusion bodies can be washed with a mild detergent, after which they will be white. They can be dissolved in 8M urea or 6 M guanidine HCl. The protein will be completely denatured and will have to be refolded, which requires experimentation to find the right conditions. Try to avoid this approach, if possible, by making every effort to express the protein in soluble form.

Adam B Shapiro you mean before i run on SDS-PAGE reduce my pellet in 8M urea or 6M guanidine HCL?just urea and GnHCL?!

what do you think if i want to run whole cell on gel?! it's not visible on gel too?!!

I don't quite understand the question, but:

1- If you want to know if you can predict whether the protein will form inclusion bodies or not: Strictly speaking no you cannot, but if it needs post-translational modifications such as disulphide bonds or glycosylation, or it uses a folding partner, or is normally part of a larger multisubunit complex, or contains a cofactor that is absent in your host, then in most cases it will form inclusion bodies. Even proteins not meeting the above criteria will often aggregate (most proteins were not selected during evolution to accumulate at high concentrations in the bacterial cytoplasm or periplasm). Also, conditions promoting rapid biosynthesis of the protein (relatively high temperatures, rapid growth rate, strong induction -autoinducing medium being one example) usually result in the formation of inclusion bodies even though the same protein, expressed at lower temperatures or poorer media such as LB, might be expressed in soluble form.

2- If you want to know whether a protein is forming inclusion bodies after expression, then lysing the cells and examining the soluble and insoluble fractions by SDS-PAGE and/or Western blotting will give you the answer most of the time (strictly speaking, you would need to analyze negatively stained cells by transmission electron microscopy to address cases of cell envelope association or particulation as in e.g. the case of viral capsid proteins).

3- You can just resuspend whole cells in Laemmli sample buffer and heat them at 100*C for 10-15 minutes. For instance, when using autoinducing medium (which yields a wet weight of approx. 0.8-1 g/50 mL with BL21(DE3) or NiCo21(DE3)) I usually take a 50 uL sample, spin for 1' at 10,000xg, resuspend in 50 uL of Laemmli buffer, heat it and load 10 uL on SDS-PAGE. Numbers will of course change for media of different productivity. If there is any viscosity left owing to released nucleic acids, a little bit of vortexing takes care of it.

Hope it helps!

In order to find out if your protein is expressed in an insoluble form, you break open the cells (such as by sonication) and centrifuge the extract. Run portions of the supernatant and the pellet on a SDS-PAGE gel. If the protein is in the pellet, then it was expressed in an insoluble form. If you wash the dirty pellet in detergent and centrifuge it again, and the new pellet is white and your protein is in it, the protein is in the form of inclusion bodies. The inclusion bodies may be nearly pure overexpressed protein. The inclusion bodies can be dissolved in urea or GuHCl. The dissolved IBs can be run on SDS-PAGE, but care must be taken. The bands will try to spread sideways because of the high concentration of salt. Make sure the adjacent wells have the same amount of solubilizing agent as the sample to keep the bands from spreading.

Thanks dear Adam B Shapiro

I just want to know I get your mean correctly! after sonication and first centrifuge, I should dissolve pellet in urea or GnHCl and after that, add loading buffer to the whole sample and run it on the SDS-PAGE gel?

would it be possible to recommend any article about procedure I should follow for running inclusion body on the SDS-gel?

Parastou Rahimizadeh ,

Do not use GuHCl, as it will interfere with migration in SDS-PAGE. Use urea instead. And yes, you did understand Adam B Shapiro 's suggestion. Sonicate, divide into supernatant/pellet, and resuspend the pellet in 8 M urea, then add loading buffer to 1X conc, heat and run it. Sometimes heating for longer than usual (say, 10 to 15 min) helps in the case of recalcitrant pellets. And of course include a reducing agent.

It is best to group the urea-containing samples into ajdacent wells, and to add a urea-loading buffer mix to the wells flanking them; otherwise they tend to expand a little bit laterally during the run and distort the migration of adjacent samples. Nothing dramatic, it just looks ugly.

Hope it helps!

Thank you very much Dr Alejandro Martin for your response,

actually I add 5x loading buffer to my sample for run in the gel?! does it differ?!

Parastou Rahimizadeh ,

Sorry, I did not explain myself very well. I meant to add loading buffer to a final concentration of 1X. The concentrated stock of course can be 2X, or 5X, whatever.