I expressed protein and unfortunately it became insoluble and remained in cell pellet. How do I get protein from pellet, so later I can use it for antibodies production?
Over-expression of a heterologous protein usually rsults in the formation of insoluble inclusion bodies. This results primarily because of the overloading of protein folding machinery of host. There are different ways of getting the soluble protein fro the inclusion bodies and one of the most commonly used method is solubilizing the inclusion bodies with guanidinium hydrochloride or urea. Once the protein is solubilized the denaturant is allowed to slowly dialyse out. The refolding conditions have to be determined and optimized for the protein in question. The extent of refolding can be measured by determining the amount of refolded protein.from a given sample.
There is lot of literature available on recovery and refolding of insoluble proteins.
HOPE THIS HELPS.
6M urea or G-HCL worked for me... I can send you my detailed protocol if you wish, let me know, but there is plenty of literature available.
Hello Prem! There are a number of detergents widely used to solublize protein. I can tell u some detergents: trition, CHAPS, sarkosyl, urea. Generally, sarkosyl works well,however it is a harsh detergent and may affect your protein activity. In case you use it try to optimize and use as less as possible to get enough protein for purification.Also make sure you remove the detergent by dialysis, so that it does not hamper your further applications.
thank you everyone for your valuable suggestions. Does it possible to run insoluble pellet on SDS-PAGE, then cut out the band, elute out and followed by the renaturation process?
@Radika kamdar, thank you. If you don't mind, Can you please send me any protocol.
6M Urea protocol worked for me very well and i was successfully able to extract protein. thanks every one.
@radhika kamdar May I get detailed protocol of purifying insoluble protein from pellet using 6M urea or G-HCL ?
Hi Prem, can you share me the protocol. i am facing same problem @Prem Prakash Das
Hello everyone, I am attaching the protocol for protein purification from the bacterial pellet. In the protocol, instead of lysis buffer use 6M urea throughout and rest of the process is same. Note: Expressed protein in inclusion body will be completely dissolved in 6M urea and rest of the protein purification protocol will be the same. After purification, dialysis will help you to reduce salt concentration.
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