Hello,
I am trying to perform immunolabelling of live cell cultures, I am looking at a secreted protein, that hopefully gets trapped at the cell surface. At the moment I am struggling with aggregations of secondary antibody that does not wash off and gives me false positive results. Does anyone specialise in this field at all? I could do with a well established protocol... any help will be appreciated.
I have done immunolabelling of live cell cultures for cell surface proteins only. I am not sure about secreted proteins. But if you are sure that they get trapped at the cell surface, then you can perform immunolabelling. For aggregations, it can be either due to non specificity of secondary AB which can be reduced by using 1% Gelatin from cold water fish skin or you are using a very high dilution. Try diluting the secondary Ab at least 10 times to set a working range by using proper diluent.
A second approach is to use fluorescent-labeled primary antibodies.
I would rather hold back from the fluorescent primaries at the moment.
How to use the Gelain please? Do I add it during the usual blocking stage? Or do I add it together with the secondary? I think this might be an option to go for.
Is the dilution of the antibody tested.....for example on the cell line at a adherent format like cell culture slides in differnet dilutions before you test them on the living ones??? This is my first idea to test your whole signaling system on methanol fixed adherent cell line on normal cell culture slides for example the type with the 4 chambers and test ditinct dilutions of your secondary antibody....after it works ther well transmit the whole system on the living ones. Where in the cell is your protein expressed is it transfered from the cytoplasma to the membrane or is it transferred from the nucleus to the ou6tside...mor details please.
Yes the dilution of the antibody has been well tested. I have used it many times on fixed cells and it always gives beautiful results. Never had any problems before. But with the live cultures I seem to see the aggregations of the antibody even without applying the primary antibody. I thought this could be blocking problem, hence I used different dilutions of the blocking agent (two different blocking agents in fact - NGS and BSA) ranging from 10%-3%. I increased the washes, but I don't want to push it in case the cells will start dying. The protein is transported from cell cytoplasm out of the cell but then it binds to the receptors on the cell membrane.
Your FBS supplement is heat inaktivated or you doing it without FBS??? Which Staining showed the fixed cells....were they whole stained or did you du a ultrasound sonification befor the staining?
We had a extracellular protein with the same problem the fixed cells showed very good stainings but the living cells showed very poor or no stainings....one thing is to longer incubation with the primary AB for example 24h in the incubator with cell media without any FBS....or to switch the secondary AB to an polymer with enhancing properties... but what me really bothers is the fact that it works well with fixed cells???
I am not sure what protocol you are refering to. They way I do it, is that I culture cells as usual (using Hi FBS), then I put the cultures on ice, wash them with ice-cold PBS, block and then put the primary antibody, wash and put the secondary antibody. Then I wash that off too and then at least fix. All the time I keep the culture on ice and all solutions I use are ice-cold. This has meant to be a nice and quick experiment but turned out to be a problematic one and I was hoping that someone might have some experience of doing this as well.
Our Protocoll was all at roomtemperature or incubation condition with a incubation time of 30min (or 24hours in the incubator) for primary AB followed from 3wash steps with at least 3min her we did a blocking with milk powder like in western blocking for 3-5min or also BSA....then another washing with PBS and a incubation with the secondary AB for 30min and the HRP reaktion with DAB....
Maybe using F(ab')2 fragment will decrease non-specific secondary binding.
Michael
Marta, you can possibly decrease your non-specific (if that is truly what it is) by removing FBS from your culture media and incubate the cells for 1-2 hours in culture media with your blocking substituted for FBS in the culture media in the incubator. The take cells out, wash on ice as you normally do. This can allow longer blocking times and better removal of non-specific binding of both primary and secondary. Of course when incubating primaries and secondaries have your blocking agent included in the incubation media as well.
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