Hi Udeshika,

There is simply not enough data to begin with - same as someone asking on an online forum what to do if his car doesn't start. there are general things to look at (and a lot written about this kind of things), but without more information, there is little chance of offering significant help. Things as simple as the protein you'd like to express and how do you know it's insoluble are missing.

D.

@ Sebastian Schmitt : Construct is totally fine. When I use other proteins to express with the same construct i observe the proteins. but this particular protein is known as a insoluble protein. In that case what should we do?

Hi! Udeshika, You can try following steps:

After sonication, renature the protein by carrying out dialysis

or,

Find out the optimum expression condition

or,

Solubilize the protein from the insoluble fraction by using surfactants

Good luck

Thank you everyone. These answers gave me new suggestions.

Hi Udeshika,

According to the current situation, you can use the method of dialysis renaturation, but I still recommend that you try to optimize the expression and purification approaches.

When purifying protein (MW does not exceed 60KD), I generally use the E.coli expression system first, because this system is economical and easy to operate, and it is convenient to find a purification protocol for the target protein in a short time. Even if it turns out that the target protein is not compatible with the prokaryotic expression system, it can lay the foundation for the next use of the eukaryotic expression system (insect or mammalian) to obtain high-quality proteins.

Moreover, you provide less information. Is your target protein a soluble protein or a membrane protein (membrane protein requires detergent during purification)? Is it a secretory protein (a signal peptide is required for this kind of protein)? Does it need to be co-expressed with chaperones or components of complexes? Are fusion tags used to help dissolve?