Reader Comments
Post a new comment on this article
Post Your Discussion Comment
Please follow our guidelines for comments and review our competing interests policy. Comments that do not conform to our guidelines will be promptly removed and the user account disabled. The following must be avoided:
- Remarks that could be interpreted as allegations of misconduct
- Unsupported assertions or statements
- Inflammatory or insulting language
Thank You!
Thank you for taking the time to flag this posting; we review flagged postings on a regular basis.
closeAntibodies against Rab14
Posted by goldenjr on 01 Jun 2013 at 17:48 GMT
Following the publication of this paper we received a note from Dr. Mary McCaffrey about our discussion about the specificity of Rab14 antibodies as a possible explanation of the differences between our results and those previously published by Dr. McCaffrey. We noted that we had tested the antibody from Abcam for cross-reactivity using 0.5 µg of recombinant Rab11a, Rab4 and Rab7 on western blots. We have used this procedure for many years to evaluate antibodies against Rab proteins, especially when they have been raised against recombinant full-length Rab proteins. The high homology of different Rab proteins makes cross-reactivity always a concern. In the case of this antibody from Abcam, we found that there was prominent cross-recognition of all three Rab proteins in addition to recognition of Rab14. In contrast, the Aviva antibody we used in this paper had no cross-reactivity with Rab11a and Rab7 and only a minor cross reactivity with Rab4. Thus, we chose to use the Aviva antibody in our studies. Dr. McCaffrey has provided to us her validation studies, which were performed with eukaryotic expressed proteins in both western blot and immunofluorescence (in Kelly, et al. 2010). In contrast to our data with the prokaryotic expression protein, they did not see any cross-reactivity. We have determined that the batch of antibody used by both of our groups was the same. We do not have a clear explanation of why there would be a difference in these screening procedures a priori. It would therefore appear that differences in antibody reactivity cannot explain the differences in our results. The divergence in the results may therefore fall to differences in Rab14 expression patterns in the different cell lines (HeLa in ours vs A431 in Dr. McCaffrey’s studies).