I am performing an IP in which I am using a Protein G-bound IgG1 antibody to bind to my antigen of interest, a glycoprotein enriched in cell culture supernatant. Once I have performed the antigen binding to the Protein G-bound antibody, I want to try its elution with potassium thiocyanate 3.5 M. However, I do not have enough sample and my elution volume will be only about 50 ul. How can I get rid of the Potassium thiocyanate from this elution volume before running them on SDS PAGE?
Hi Bhavana, use 8M urea instead of KSCN. K+ forms an insoluble complex with SDS.
Thank you for your answers!
@Ziguo Zhang: I checked the Zeba spin desalting columns Ziguo Zhang. Could you suggest one more thing if you have used these before? It says in the protocol that "Apply a 3µL stacker of ultrapure water or buffer to the top of the resin bed after the sample has absorbed." Is it better to use some buffer instead of ultrapure water? If so, what buffer?
@Steingrimur Stefansson: I have used 8 M urea but I'm unable to elute my protein out, which is why I am trying harsher methods of potassium thiocyanate and guanidine hydrochloride. I was just wondering if we can dilute it in a way that it makes the sample possible to load on sds page? But I cant dilute too much either because I may end up detecting nothing on the western blot. Is it possible to exchange the KSCN with 8M Urea?
Hi Bhavana, try boiling the sample in 8 M urea with 0.1 SDS. If there is nothing coming of the beads, then I a am afraid there is nothing bound to them.
Also stay away from Guanidinium-HCl, it will also precipitate with SDS.
Okay thank you I will try boiling with 8M urea to see if something is eluted. Yes I know about Guanidine HCl but I found a protocol on another question in research gate describing about how I can manage to run a sample by avoiding the precipitation of that. So I will give it a try. Thank you so much!
- Badges
- Science method