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closeHow is it possible to detect active GTP-bound form of Rap1 by Western blotting?
Posted by ostep on 25 Feb 2014 at 14:10 GMT
Wittchen et al. used an antibody recognizing active GTP-bound form of Rap1 (Figure 5D). Actually, this paper is the only reference to that antibody on the manufacturer's (NewEast Biosciences) web page: http://www.neweastbio.com...
Rap1, as all GTP binding proteins, bind GTP/GDP non-covalently. This means that denaturation of GTP-Rap1 by SDS in standard Western blotting protocol (SDS-PAGE and immunoblotting) should result in the release of GTP from Rap1. I am very surprised that authors claim that they were able to detect active GTP-bound form of Rap1 by Western blotting (Figure 5D). One possible explanation is that they could have used a kind of native electrophoresis prior to the immunoblotting, but this is not specified in Methods.
The manufacturer recommends to use this antibody for immunoprecipitation and immuno-histochemistry. The respective datasheet ( http://www.neweastbio.com... ) includes an experiment, where untreated and GTP-gS treated Rap1 solutions were first immunoprecipitated with the antibody to active GTP-bound Rap1 and then the amount of pulled down GTP-bound Rap1 was analyzed by Western blotting using an antibody to total Rap1, which does not discriminate between the forms. If such an easy method (Western blotting) worked, why would the manufacturer describe a longer two-step protocol for showing that the antibody works?
Another related question is, why the authors did not measure GTP-Rap1/total Rap1 ratio, but GTP-Rap1/actin ratio instead. Is it possible that the GTP-Rap1 antibody actually stains total Rap1 in Western blotting and the treatment shown in Figure 5D just increases total Rap1 levels?
I believe that this issue should be clarified not only in respect to the conclusions of the article by Wittchen et al. It could also help to set the methodological standards to analyze active GTP-bound forms of GTP binding proteins.
Dr. Ondrej Stepanek, University Hospital Basel, Switzerland
RE: How is it possible to detect active GTP-bound form of Rap1 by Western blotting?
ewittchen replied to ostep on 28 Feb 2014 at 16:12 GMT
Dear Dr. Stepanek,
Thank you for your interest in our paper. We have used the GTP-Rap1 antibody (NewEast Bioscience) to detect active Rap1 in retinal tissues both by western blotting (this paper), and by immunoprecipitation as recommended by the manufacturer in a follow-up paper (FASEB J. 2014 Jan;28(1):265-74).
Under both conditions, we obtained the same result that intravitreal 8CPT-2’O-Me-cAMP injection increased Rap1 activity in retinal tissues. Importantly, in the second study, neither laser injury nor 8CPT-2’O-Me-cAMP treatment affected the total Rap1 levels. Therefore, we believe using the ratio of GTP-Rap1/β-Actin does not generate a significant difference from using the ratio of GTP-Rap1/total Rap1 in evaluating Rap1 activity in retina.
For our experiments, we used NuPAGE Bis-Tris precast gels, which, according to the manufacturer, have a neutral pH environment that minimizes protein modifications and can be used for sequencing, mass spectrometry, and any other application in which protein integrity is crucial. It is possible that this may account for the detection of GTP-Rap1 in our Western blot samples.
Thank you for the opportunity to clarify our findings.
Sincerely,
Erika Wittchen, PhD
Haibo Wang, MD PhD
Mary Elizabeth Hartnett, MD