Sorry Rao, but I have no idea what "IB solubilisation" means for you. BME is a monothiol, DTT is a dithiol reagent. But what is your problem? What is your aim? I cannot figure out your clear question or problem.

Thanks Dr Alajos,

I am targeting a E.Coli expressed protein solubilisation and purification by Hist tag.  The target protein is expressed and  purified in denaturing conditions and the yield is also good. My target is protein in SOLUBLE form. In the above process to increase the solubility I am trying alternative methods of inclusion bodies (IB) solubilisation. Protein Does not also need any folding. In the IB solubilisation I am planning to work either with DTT, BME, Sodium DeoxyCholate subsequently to dialyse them against Tris buffer and IGEPAL 0.2% for their removal of reducing agents.

Kindly advise any ideal or optimal conc of the above from your expertise.

Buffer under the trial prefered is Tris 50 mm pH: 8.0 10% Glycerol, 10 mm Imidazole.

Kindly advise and if any additional information required may please be mentioned.

Thanks in advance,

Regards

I am sorry but I have never ever worked with E. coli or E. coli expression systems. I work with yeast expression systems. So I have no experience with IB or its solubilization, etc. Furthermore, your target protein is either a soluble one or a membrane-bound one. For soluble proteins you do not need detergents for purification although due to other reason than solubilization mild detergents can be added at some steps in the purification.Working with detergents needs some idea why and for what you want to use a detergent. Proteins which can be purified only after solubilization with detergents will require solutions with detergents present at concentration of higher than its c.m.c. throughout the further purification steps. Detergents without c.m.c. cannot be used for protein solubilization but can be employed as surface active agents.

Since the reducing agents are small molecules, I prefer to remove them by a SEC step using a PD-10 column, if needed; it is much faster than dialysis and needs much less detergents, if it was included.

During the homogenization step (or tissue destroying step) I use a few millimolar (

We have successfully purified inclusion body protein, but the yield was poor, about 10%. Most of the protein was lost when concentrating. Most of the information is in the attached reference & supplement published in Vaccine.

The main purpose of adding a reducing agent during purification is generally to stop unwanted protein aggregation. Proteins you are not interested in (and which are therefore classed as contaminants) can bind to your protein of interest via disulphide bonds. Adding a reducing agent stops this from happening. The choice of reducing agent depends on your purification method. In your case the protein of interest has a His-tag, so I guess you're using some kind of Ni-NTA, which means you need to be careful as a strong reducing agent will not just reduce the disulphide bonds of your protein but also the nickel ions of your resin, which results in a brown mess. Most nickel resins can withstand up to 5-10 mM BME (which is the usual choice for Ni-NTA as it is a weak reducing agent), but some can also be used with 1-2 mM DTT. DTT has the advantage of being a longer lasting reducing agent, but since most people remove the BME straight after Ni-NTA, it doesn't really matter that BME only works for a maximum of a few days.

I've read that guanidine-HCl seems to be the denaturant of choice for most people's IB solubilization. DTT is quite often used in the 20mM range. If you are going to later use IMAC, why not just dilute your sample out in binding buffer until you reach the acceptable DTT or BME concentration?