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Blocking a protein in cell culture through antibody

blocking inhibiting antibody

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10 replies to this topic

#1 Tabaluga

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Posted 18 August 2013 - 12:35 PM

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. unsure.png  Has anyone got an idea ?

Thanks !


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#2 Curtis

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Posted 18 August 2013 - 07:25 PM

do you have FBS and antibiotic in media during incubation?



#3 Tabaluga

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Posted 18 August 2013 - 08:09 PM

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 ?


Edited by Tabaluga, 18 August 2013 - 10:04 PM.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#4 Curtis

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Posted 19 August 2013 - 12:15 AM

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.



#5 Tabaluga

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Posted 19 August 2013 - 12:44 AM

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.


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#6 science noob

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Posted 19 August 2013 - 04:15 AM

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. unsure.png  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?


Edited by science noob, 19 August 2013 - 04:17 AM.


#7 Tabaluga

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Posted 19 August 2013 - 04:19 AM

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.


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#8 SerAngie

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Posted 30 October 2013 - 03:44 AM

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!!!



#9 Tabaluga

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Posted 30 October 2013 - 04:37 AM

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...


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#10 SerAngie

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Posted 30 October 2013 - 10:36 AM

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.



#11 Tabaluga

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Posted 30 October 2013 - 12:26 PM

Hi, yes, of course I know that the antibodies I tried were all against the extracellular domain. I don't know a lot about permeabilization, but a quick google search for "permeabilize living cells" gave some results so maybe it's possible, but I would not recommend it as it could interfere with a multitude of processes and affect your blocking results.

The more obvious way would be to buy an antibody against the extracellular domain of your membrane receptor (or is no such antibody available?).


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 






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