I'm studying the localization a GFP-tagged protein in arabidopsis roots, but since this protein is very dynamics and degrades very quickly, I'm considering fixing my samples.
I assume that the GFP fluorescence will be compromised due to the fixation, so I was thinking on using an anti-GFP antibody. However, there are commercially available specific antibodies against my protein of interest.
It would be better using specific antibodies for my protein or anti-GFP antibodies? What gives more reliable information?
Thank you for the suggestions :)
Dear Liliana Ferreira ,
You are correct. There are some fixation methods that can hamper your signal-to-noise ratio and increase background noise. This generally occurs with the application of aldehyde-based fixation methods. You may treat your sample with glycine or ammonium chloride to minimize those effects.
Yet, by conjugating GFP and an anti-GFP antibody you can minimize those effects even more – it is something you should observe and test in your experiments.
If you apply an antibody for your tagged protein you will have two competing fluorescent signals (i.e. GFP + the signal from your antibody targeting your tagged protein), what can be unwanted, as you would merely be assessing, in confocal microscopy, how both of these signals truly colocalize.
Nevertheless, if you just want to identify your tagged protein in the tissue, and amplify/specify its signal, I would reccommend using an anti-GFP antibody (conjugated or indirectly reacted with an Alexa Fluor 488/FITC fluorophore/Ab). But be sure to check whether your protocols leave you with such a noisy image with the GFP tagged protein alone if you can.
Hope this helps.
I have used a 4% formaldehyde solution for fixation before and had no trouble with detecting GFP fluorescence in my fixed cell samples. The signal might be weakened by the fixation, but I think it is worth a try before buying expensive antibodies.
In case you still want to use antibodies, be aware that AlexaFluor488 and GFP have overlapping emission spectra.
Good Luck!
Dear Liliana Ferreira
I have used 4 %PFA fixation with GFP tagged proteins and it worked well for me. However, if you are not getting good GFP signal upon fixation, you can go for using specific antibody for your protein of interest. But in this case, you do not need GFP tag in your protein.
If you are using GFP tagged protein, then try using secondary antibody having different emmision spectra other than Alexa Flour 488. You can use Alexa Flour 555 which will produce yellow/ orange signal upon overlapping with GFP signal and you will eventually land up with a good image with less background.
Hope it helps.
Good luck!
Thank you very much Eduardo Listik, Jan Philipp Dobert and Bindu Singh for your help :)
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