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closeAntibody selectivity
Posted by norbertg on 26 Nov 2014 at 17:24 GMT
The Abcam antibody #ab29112 is directed against amino acid 1001-1054 of SALL4A (http://www.abcam.com/sall...) while the Abcam #ab57577 is directed against amino acids 953-1054 of SALL4A (http://www.abcam.com/sall...). These 2 antibodies are thus directed against overlapping epitopes located in the very C-terminus of SALL4A.
SALL4B lacks amino acids 385 to 821 of SALL4A, but still contains the epitopes against which ab29112 and ab57577 are directed (http://www.uniprot.org/un...).
Using the antibodies referred to by the authors in Materials and Methods, it is not possible to discriminate between SALL4A and SALL4B isoforms on immunohistochemistry as proposed (figures 1 and 2).
RE: Antibody selectivity
korwig replied to norbertg on 31 Dec 2014 at 22:07 GMT
Thank you for your comment. We are reviewing our original antibody validation data, the product information sheets and our IHC data. The Western blot data in figure 1B clearly show a shift in SALL4 isoform expression during early postnatal development that corresponds with the IHC data in Figure 1A. However, we acknowledge that the interpretation of the IHC data is suspect since it appears that both antibodies were raised against similar epitopes, albeit from different species. We are trying to ascertain what led us to believe that AB57577 was specific to SALL4A. Please note western blot images from these two antibodies in the product information sheets. AB57577 reveals only one band in HeLa cells and the company does not speculate what that band might represent. No mouse WB data were provided for this Ab in the product information sheet and we are reviewing our own notes to see if we validated the Ab by western blot using mouse tissues. Meanwhile, we are currently testing both antibodies by Western blot using lysates from mouse testis cells (focus of this paper), human HeLa cells, mouse embryonic fibroblasts (negative?), mouse ES cells (expressing both isoforms) and cells expressing only SALL4A or SALL4B from expression vectors. We will comment again once these analyses are complete.