Membrane proteins require detergent to keep them in solution when they are not in a lipid membrane. Once the urea is washed out, the protein will precipitate if there is no detergent to keep it in solution. Some commonly used detergents are CHAPS, n-octylglucoside and dodecylmaltoside, although there are many others.

Adam B Shapiro Thank you for the answer, sir! These detergents seem to be expensive.

0.5 M urea will not denature anything, check for conditions using cheap surfactants such as tween-20.

There are certainly less expensive detergents, such as Tween-20 (mentioned by Omar Gonzalez-Ortega), Triton X-100, Brij-35 and many others. However, if you are going to use polyoxyethylene detergents such as these, you should be careful about their quality, because they oxidize in air into peroxides that can damage your protein. When I use them, I buy them in 10% solutions in glass ampules under inert gas, which obviously increases the cost substantially. Alternatively, buy a new container, prepare 10% solutions in distilled water, and store them in the -80 freezer.

Full denaturation by urea usually requires at least 6 M, if you are starting from inclusion bodies. Guanidine HCl can also be used.

Dear Dr.

The problem is low protein production or low purification efficiency. Of course, variables affecting production have many interactions with purification. If the increase in protein production does not lead to the formation of the desired form of protein or if more than 90% of the produced protein is not transferred from the cytoplasm to the membrane for some reason, the purification efficiency will not increase despite the increase in production. Consider the hydrophobic and hydrophilic properties of protein in choosing the right detergent. Maybe measuring the total protein next to the electrophoresis gel can determine to a great extent in which of the pure steps you lose a lot of protein.

Hi,

Is the Ni-resin OK?

Have you ever regenerated the resins with Nickle salts?

Is it possible to add another step before affinity chromatography?

Is it possible to try with other Ni-column?

Is it possible to use Ni. resin in batch method instead of using column?

Dina Morshedi I have regenerated the resin with NiSO4 whenever I felt the resin performance has been decreased or change in color of the resin is seen.

Prathyusha Thogiti, Valiollah Babaeipour , higher protein production does not necessarily mean higher protein yield during purification. Increasing too much production may actually result in higher toxicity for the recombinant host, as well as higher precipitation rates, leading to a lower yield in proportion. So aim for good quality before going for high quantity.

That said, there are a number of factors that can influence both:

- expression host

- expression conditions (medium, additives, induction method, time, temperature, etc)

- construct, tags

And then yes, as said above, membrane proteins do require a lipid environment, if you remove that, then your protein is not going to be happy

Dear Prathyusha Thogiti

membrane proteins are generally toxic for the cells and therefore it is not easy obtain high yields. HOwever in your case is important to undestand which is the factor that limit your yield:

1)Is the expession level that is low? And this could be estimated from SDS-page or WB of total extract;

2) Is the recover low? You have low extraction efficiency or low recoved during purification?

Because if the prolblem is the first i think that you cah just try to scale up your culture volume or use a media are the Enpresso which allow to produce high biomassess.

If the problem is the extraction and or purification, as Adam suggest you have to replace Urea with other extraction agents (eg detergentes as DDM, DM, OG) which are probably more expensives but more efficient for a membrane protein

in case you are interested in the following link you can find more information about exnpresso media

https://www.blogger.com/video.g?token=AD6v5dwU8Sl-43BfVTSeIRFBcQiWXAeg80Ud5UCxiXqUj4AFUO7p1BOHHmV4NiV0MY32A41mTgUH2-nyouDVBhcVFOuGc1lJ5_1WgP3RrsvakB6iJHCW_JOnrD8kIeiUSimWYahOs-OP

best regards

Manuele