I'd like to investigate if my protein of interest is covalently bound to the protein which was caught by the antibody. Can I leave the elution buffer (which is my SDS sample buffer) without DTT? Or has DTT some effect on the elution?
I want to save the "covalenty" disulphide bonds and see if my POI is bound via a disulphide bond.
It will definitely work w/o DTT.
You could also try reducing your sample before IP and then add iodoacetamide to protect cysteines from reoxidation. This iodoacetamide will also inactivate DTT towards reduction of the antibody. Control for affection of the IP by iodoacetamide would leave out the DTT. If only the control pulls down your POI it is a strong indication for a co-precipitation by disulfide linkage.
So I tried it using SDS Loading buffer without DTT and I got a "nice" elution, but with functional IP antibody, so hm... I now try to optimize my elution using glycine + low pH or high pH and encounter with the respective, or ion strength elution, like 3 M KCl. Thank you for your help!
At the end, it worked (kind of) without reducing agents, SDS and LDS worked well. In my case it elution efficiancy was dependend on experimenter's harshness; always resuspend beads as good as possible and elute with harsh shaking. Be kind of rude!
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