KLIK

Hello I am working on cytotoxic T-cell assays. Iam planning to use dendritic cells pulsed with recombinant proteins as a stimulators in Cytotoxic T-cell assay (CTL assay). I have used particular cell lines (tumor cell lines) as stimulators in all the CTL assays that i have done so far in which i used to treat the tumor cells with mitomycin C before using them as stimulators. Can i follow the same procedure with pulsed DCs also. I have tried to find specific protocol for using pulsed DC s as stimulators but all the references i have gone through just mention this in few words but not any clear procedure. Can anybody help me with this or u can tell me references in which i can find the clear and complete procedure. I appreciate ur help very much.

Thanks for your explanation

hey Thanks for your suggestion. Can you please tell me little bit more about CD (circular dichroism) spectrum. Iam not at all familiar with this.

when i said "HSP70 as a part of it" i mean to say that my gene X ( produce protein X when expressed) of interest was fused to HSP70 and the final protein obtained was recombinant fusion protein X-HSP70.

Thanks for your reply. I dont have access to NMR (well our department is not well equipped).i dont have much experience with protein chemistry so I read in some of the protein purification protocols books - it was said that when protein is purified under denaturing conditions with urea, removal of urea by dialysis will bring the protein to native state. so using the procedure that i mentioned before i was trying to do that. My experiment is to use the pure protein for immunizing mice and look at their immune response. Anyway i will try to get some help NMR from someone. Thanks again for your suggestions.

Hello Iam working on recombinant fusion protein which has HSP70 as a part of it. (This protein has His-tag too). protein was expressed in bacterial cells and purified using Nickel column under denaturing conditions (8M urea). In order to refold the protein after purification elute fraction (protein eluted with 8M urea pH 4.0) was dialyzed against 10mM Tris (pH 8.0), 0.1% Triton X-100 followed by dialysis in PBS (pH 7.4). Does this procedure refold the protein or not? I need some help in this matter. can anybody answer my question. i appreciate your help very much.