How do you know that your antibody is not picking up the correct protein? Do you have a control?  Have you run the sample under both native and denaturing conditions?  Do you see a shift in the apparent band size?  How can you make any inference about the expression of the protein before you know if your detection method is working?

Dear Marcus,

I did not say that my antibody picked up a wrong band, rather it is also showing some nonspecific bands.

Secondly, in SDS I can definitely see an induction but it was not convincing.

The plasmid has the insert for the protein with His tag and the expected molecular weight is around 50 kDa. If you receive a signal at 50/51 kDa probing with an anti-His antibody, don't you think that you are getting a positive result?

But, that signal is not enough convincing to go for further purification step? 

I appreciate your comment but unfortunately, it is not helping me at all.

Cheers

I apologise profusely, I completely missed the 'along with the band of interest'!

The key point here is that you have a poly-clonal anti-His tag antibody. There will be other histidine-rich proteins in the cell that it will react to.

You just need to purify the protein with IMAC and test if the non-specific bands disappear in Western.

Alternatively, get a monoclonal anti-his antibody or even better, if available, a specific monoclonal antibody against your protein of interest.

Your other problem seems to be one of protein yield. Generally, in a bacterial expression system, if you can only detect your expressed protein by Western and if you can't see the induced band by just staining (compared to an uninduced or no insert plasmid control), then you have an expression yield issue. Your approach to optimisation by varying [IPTG] and temperature is a sensible one. In the first instance, I would blast it with 1-10 mM IPTG and check induced Vs control on SDS PAGE/Coomassie just to see if the protein is able to be expressed at high levels. Check where it is - soluble or inclusions? If soluble, you are in luck. If inclusions then play with IPTG, temperature, glucose suppression etc.

In the light of your comment to Marcus, I sincerely hope that my comment helps you somewhat!

Hi Abir,

decreasing IPTG concetration and expression temperature are two widely used strategies to help proper folding of recombinant proteins. So I think you are already going into the right direction. In this regard you might want to consider using an auto-induction medium (I attached the paper), which gives a more gradual expression and it could be helpful. Unfortunately this does not imply necessarily that disulfide bonds will be properly formed. You are already using a strain engineered to improve it, but maybe you could also try to target your protein to the periplasm.

This is all I can think, I hope it may help. Proteins with many disulfide bonds are notoriously hard to get in E.coli, have you considered alternative expression systems?     

Dear Patrizia,

Thanks a lot for your comment. Yes, I have a plan of doing in the  Pichia system. 

 Dear Deepan, Yes! your comment is helpful. I am probing with the protein of interest now with a positive control as well just to confirm that the antibody is working.Thanks a lot.

Not sure if it will help, but I use 10-20 mM DTT to avoid di-sulphide bond formation of my protein. 

Why should I avoid disulfide bonds where these are crucial for that protein? 

Thanks for your comment.

I just thought if your protein has cysteines then it may form dimers and oligomers, may be that's the reason you are seeing your protein at higher MW on your western. I may be wrong but my protein has also cysteines and they form dimer or oligomer until I add DTT during protein purification (nickel or size exclusion) and then it shows correct MW on SDS gel.

Hi,

I have also observed some higher molecular weight band only at the un induced sample. After two hours post induction that higher molecular weight band is not picked up by the antibody specific to the protein of interest. 

Dear Abir Chakraborty,

Maybe your protein in inclusion bodies is not recognized by the antibody. Some higher molecular weight band seem to be aggregates. Have you already  tried to run a reduced SDS-PAGE before transfering to the membrane of Western blotting?

We used to add 1 mM IPTG with cultures at 30 °C, 32 °C, 35 °C, 37 °C and 42 °C in order to find the best temperature for a new protein expression.

You can consider to express the protein into the periplasm by using another vector as pET39b(+) with DsbA (disulfide bond A) as signal peptide that can improve disulfide bond formation.

Good luck!

Miriam

Dear Abir

Professor Lloyd Ruddock´s group, in which I am working, has made a technology which helps in the formation of disulfide bonds. It is better then shuffle. CyDisCO is the name of technology. And has successfully produced protein which has 22 disulfide bonds. You can also consider this technology for complex proteins having lot of disulfide bonds. 

Dear Anil Allan Sohail,

May I have that protocol?

You can email my professor with my reference and tell him your problem. He might be able to help. 

Thanks a lot. Sorry for a stupid question. Is this CyDisCo an E.coli strain? I will definitely contact him

Please see the link for details. i hope it will help.

http://www.oulu.fi/biocenter/groups/ruddock

At what Mw is running this high molecular weigth protein? in some cases, proteins with high helicity run at a molecular weight which is different to the theoretically calculated weight regarding a globular standard protein. This highly anomalous membrane protein PAGE migration behavior may be due to the hairpin conformation and therefore an altered detergent binding (Rath et al., 2008). Sometimes, for specific proteins you can also obtain multimers even in SDS-PAGE.

f you have obtained in your western blot a clear mark in your positive induction samples and not in your negative induction samples with your Ab, the interaction should be specific.

Regarding expression conditions, I would clarify the previous part before changing conditions. Just try standard condictions (37ºC and 1 mM IPTG) to see a clear induction, afterwards you can move to different IPTG concentrations and temperatures in order to obtain a pretty folded and soluble protein. If you begin decreasing too much the temperature, you can loss expression

Dear Ana,

I did get a clear consistent band with my protein of interest when I probed with the protein specific antibody and this time there is no additional non-specific band. Unfortunately, II am getting an another problem in the purification step.I found my protein in the pellet but not in the supernatant which I think it is inclusion body bound proteins and I need to try help solubilize with the Sarco Syl.

Dear Abir,

Then, maybe you should try to change culture conditions (IPTG and temperature) in order to improve the solubility of your protein, or other strategies such as GST-tag fusion or a different heterologous expression system.

Dear Ana,

 I have finally optimized the temperature and IPTG concentration. Expression profile is very much accepted but now the problem is the purification. It is not coming in Sup, rather it is stuck to the pellet. I have decided to use sarcosyl to dissolve the protein. I have resuspended the pellet in sarcosyl solution and kept at 25 C for proper solubility. I did not put at 4C considering the temperature and the rate of solubility at a lower temperature. Do you think I am doing right or wrong?

It can be a copurifying chaperone. I would definitely send it for MS. Unfortunately this can sometimes mean a not properly folded protein of interest. But perhaps not. The chaperones can be removed by different protocols with ATP protocols (in combination with high salt, detergent or a little urea) But sometimes they stick phenomenally strong to proteins (especially HSP70).

The CyDisCo system is an excelent idea. I would try it in combination with His-MBP with TEV site linker. There are special protocols for TEV cleavage with glutathion reducing agent that are compatible with disulphide bonds.