Hello Friends!
I am doing protein expression in E. coli (BL21 (DE3 CC). I want to extract and purify protein from bacterial culture. I need your suggestions and your experience for protein extraction and purification. Can any one tell me which method and which kit is the most efficient for protein extraction and purification.
Kindly guide me so that i can do that without wastage of resources.
I need protocols for both soluble and insoluble proteins.
You need to go through the GE manual on "Strategies for Protein Purification" for a successful protein purification. It discusses all the if and buts.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368116/
https://www.slideshare.net/chemist780/techniques-for-protein-purification
Hello Hafiz,
Since your protein is His-tagged, it is better to start with a Ni-NTA column (Affinity purification). So after you have done induction studies and established the various conditions resulting in a high expression of the protein of interest, it is better to do a small scale purification. First of all you need a a suitable buffer. In that case check the pI of your protein. Then you can choose the appropriate buffer of apt pH(+/- 1). For my protein (pI = 5.7) I use tris buffer at pH 7.0. Then after sonicating the cells to release your protein, you need to separate the sup and pellet. Assuming that your protein comes in the sup, bind that with the Ni-NTA beads, pass through silica gel bead column and elute out your protein with a imidazole gradient. Run it in a gel to check whether another step of purification is needed or not.
This link is going to help you more https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909483/
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