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Time and Temperature: antibody incubation in Immunofluorescence - (Sep/02/2013 )



I would like to know if there is a difference between the time and temperature of the antibodies incubation during IF protocol, e.g.:


Primary Antibody: 1 hour in room temperature or 4*C overnight? 

Secondary Antibody: 2 hours in room temp. or at 37*C?


This time, I incubated the first for 1 hour in RT and the second for 2 hours in RT too. When I saw the result, the second antibody fluorescence was too low and I could only see the bright cells using a 40x magnification.


My goal is to see if 2 transfected proteins are co localized, but the problem is that the "protein A" has a GFP tag (pEGFP-Protein-C3)  and the protein B, an HA tag, forcing me to use a primary and secondary Ac. 


The GFP tag work beautifully but fight with glow of the secondary Ac. Even if I want tho see a co localization, in this way, I will only see The Green! 


What do you guys think?


I will appreciate any help! 


Thank you!



The incubation times have little to do with the amount of fluorescence you see, so long as you leave the antibodies on long enough for maximal binding, the fluorescence signal is more due to the abundance of the protein of interest, the colour you are looking at (e.g. green appears brighter to the eye than an equal amount of red), and the distribution of the protein - localized concentrations may be more easy to see than a diffuse staining.


The incubation times and temperatures are more about specificity - as the temperature increases, the antibody binding gets less specific, so you should use a shorter time to prevent non-specific bindings.  Secondary antibodies should be used for the minimum time possible, as these are most often the source of non-specific binding and hence background signal.


When you are taking the images of your cells, you should be able to adjust exposure time and use of neutral density filters so that your GFP fluorescence is not too bright, then have different settings such that your other signal is brighter.  If you have chosen your secondary antibody and filter set properly, there should be very little signal from the GFP in the other channel, likewise for the other signal in the GFP. 


Thanks, Bob! You're the best! biggrin.png