Hi,
I am growing my cells in M9 media for P450 protein, but my heme is not binding with my protein after purification.
Please help with this.
Such problems are quite common when any body works with p450. The best way is to repeat the experiment and check which of the components of M9 medium is the culprit. I feel that it is the Iron source which is the culprit. Try reducing it, may be you will get a better result. Similarly it is with the phosphate concentration also. These 2 mainly interfere with the binding of heme with the protein.
Since it is expected that apo-P450 is normally capable of binding heme added to it, a possible reason for the lack of binding in your hands may be that the P450 is not folded correctly, i.e. it is denatured. It may also be aggregated or proteolyzed. Look at your purification protocol and see whether there are any points at which the protein might have become damaged.
Since P450s are membrane-bound enzymes, you must have used a detergent to extract it and keep it in solution. What detergent did you use, and at what concentration?
Hello Adam,
Thank you for your reply, I am using ammonium sulfate for extraction and I am using HIC Column for purification.but people are using same procedure in my lab for normal LB (Luria broth) culture they didnt get any issue with this. I dont know if M9 has any important role in this for not binding the heme.
- Badges
- Science method