Blocking a protein in cell culture through antibody - (Aug/18/2013 )

Hi everyone,

 

I have been trying for ages to block a surface protein on my cells through administering a specific antibody that binds to the protein and thus inhibits physical interactions with other proteins or the same protein on other cells. However, I never had success - I never see the expected morphologic effect. I've so far tried:

 

- different antibodies

- different antibody concentrations

- different scales (cell densities and well plates)

- different ways of administering the antibody (adding directly to cell culture vs. preincubation with subsequent culturing)

 

The one thing I have not yet tried is to preincubate, culture and add the antibody again every 24 hrs or so. But I don't think the antibody is degraded so quickly when the cells are in medium, for I have found that it is detectable by staining flow cytometry even days after administration. I should also add that I had proper controls (isotype and no antibody), and the antibody-treated cells look like the control cells.

 

I am at a complete loss why it is not working or what I could try next.   Has anyone got an idea ?

Thanks !

do you have FBS and antibiotic in media during incubation?

No FBS, as the cells I'm working with are stem cells and have to be kept without FBS. But yes, I do have Pen/Strep in my medium. Why, do you think it interferes ?

I think usually this kind of research should be in media without FBS and antibiotic, at least until the antibody binds to target. Later you can replace media and add fresh. My friend used to this type of research, but he failed because the membrane protein didn't exist on the cell at all. He wasted nearly a year on that, then realized the people who gave the cells to him made the mistake.

Interesting point. But when I had preincubated the cells with the AB before culturing (I did it like with a normal FACS staining, except that I didn't add a secondary AB), the cells only came in touch with antibiotic-containing medium after it the AB had bound, as you have suggested. I can give it a try though next time, culturing completely without antibiotic.

Sorry to hear about your friend's experience. I've been at this for a long time now too, but I know for sure that my cells do express the relevant membrane protein.

Tabaluga on Sun Aug 18 20:35:31 2013 said:

Hi everyone,

 

I have been trying for ages to block a surface protein on my cells through administering a specific antibody that binds to the protein and thus inhibits physical interactions with other proteins or the same protein on other cells. However, I never had success - I never see the expected morphologic effect. I've so far tried:

 

- different antibodies

- different antibody concentrations

- different scales (cell densities and well plates)

- different ways of administering the antibody (adding directly to cell culture vs. preincubation with subsequent culturing)

 

The one thing I have not yet tried is to preincubate, culture and add the antibody again every 24 hrs or so. But I don't think the antibody is degraded so quickly when the cells are in medium, for I have found that it is detectable by staining flow cytometry even days after administration. I should also add that I had proper controls (isotype and no antibody), and the antibody-treated cells look like the control cells.

 

I am at a complete loss why it is not working or what I could try next.   Has anyone got an idea ?

Thanks !

 

Have you tried non-antibody inhibitors for your cell membrane protein? Maybe something like a chemical inhibitor or RNAi against your protein of interest?

These could be other ways you could block or silence your membrane protein.  Or what you can try is find out if there are any specific binding partners (ligands) for your receptor?

Thanks for your comments. Chemical inhibition would have been our preferred method, but there is no known specific inhibitor for our protein. And knockdown is unfortunately not within the scope of my project - it will be performed eventually by a colleague, but it's still ages away, and for my thesis project I have to get along without.

Hello Tabagula,

 

I was wondering if you could solve this issue since I am planning a similar experiment. Could you achieve the blockage? Do you have any advice? I am working with Xenopus laevis oocytes and I wanna inhibit a pathway blocking its membrane receptor with the specific antibody. 

Any tip will be very welcome!!!!

Thank you!!!

No, unfortunately I didn't have any progress. In the meantime I have seriously began to doubt that the "expected" effect would take place even if the inhibition was taking place (which it perhaps is, we don't know for sure). Maybe in our cells for some reason the protein is not involved in this effect.

Sorry to be of no help here...

Thanks Tabaluga... checking the datasheet of my antibody I realised that it was designed against the intracellular domain of the receptor I wanna block...so, sadly I want be able to do it...I will try to find a protocol that allows me make a bit permeable the cell to my antibody without affecting my system...I am not very optimist but I'll try. 

I am sorry if this is very obvious for you...have you check if the region recognisable for the antibody is exposed in your system? here in my lab they have done this technique in a different cell type and they have succeed. They only did an additional blocking step for avoiding unspecific binding sites and after a few washes they placed the cells into the culture media with the antibody. Hope this tips can be helpful.

Thank you again and if you have any idea about making permeable cell membrane without affecting too much the system, I will really appreciate any contribution!

cheers.