The mRNA binding protein FOX3 (aka Rbfox3, Hrnbp3, A6nfn3) is now well known to be the same protein as NeuN, the "neuronal nuclear marker", first described by Mullen et al. in 1992. A mouse hybridoma was cloned out because it produced a monoclonal antibody, called A60, which had an interesting and potentially useful neuron specific staining pattern. The mouse had been given a very unusual mixed antigen immunization protocol and the protein target of A60 was unknown. They noted that the A60 antibody bound to a doublet of 46-48kDa on CNS homogenates but also a weaker band at about ~70kDa. The A60 antibody was subsequently licensed to company now owned by Millipore-Sigma who have marketed it for many years, see here. In 2009 Kim et al. identified the NeuN protein as FOX3, one of the very large family of RNA binding proteins. The 46-48kDa bands are due to alternate transcription producing four different protein variants. By immunoprecipitation and mass spectroscopy they showed that A60 also binds synapsin 1, but with about 20 fold lower affinity compared to binding to FOX3. Despite this cross-reactivity they showed that A60 did not bind synaptic regions rich in synapsin 1 in stained sections. They showed that epitope for A60 was within the N-terminal 106 amino acids of human FOX3 using recombinant constructs. These authors pointed out a proline rich sequence in synapsin 1 is similar to but not identical to sequences at the N-terminus of FOX3. Based on this similarity they proposed but did not prove that the A60 epitope was at the N-terminus of the molecule, the region of homology with synapsin 1. Later we mapped the exact epitopes for both A60 and our own monoclonal antibodies to the center of the peptide YPPAQYPPPPQNGIPAEYAP, amino acids 5-24 of the human sequence, which is the region Kim et al. had postulated to contain the A60 epitope based solely on the synapsin 1 sequence homology. Our work firmly maps the epitope for A60, MCA-1B7 and several other of our FOX3 monoclonals epitope for the first time (see here for details). The proline rich peptide proved to be recognized by 5 different monoclonal antibodies made against human FOX3, so this region clearly must be unusually immunogenic. We also showed that our antibody is more robust than A60, showing a higher titre and stronger kinetics of binding, and this data is on our web site. Mao et al. 2016 studied the synapsin 1 cross-reactivity of A60 in detail. Their figure 1 shows weak staining of synaptic regions in rat hippocampus with the original A60 antibody, but much less with the same antibody licensed from Zymed, a company now part of ThermoFisher. This is of course a surprising result. They used VGLUT1 and synapsin I antibodies to label these same synaptic structures identifying them as mossy fibre terminals. Their figure 4 shows a western blot of A60 on hippocampus and cortical extracts revealing a band at about 70kDa along with the expected 46-48kDa doublet. They provide compelling evidence that this band corresponds to synapsin 1, since the western blot image obtained with synapsin I antibody overlaps the A60 band perfectly.
The presence of another band at about 70kDa has been noted by many other workers, both using A60 and other antibodies raised against recombinant or peptide sequences of FOX3. Here is a rather awful blot from the very expensive and supposedly well respected Cell Signalling Technology here, showing a mysterious extra band at ~140kDa. This one, from Bioss has much stronger staining for the ~70kDa band than it does for the 46-48kDa doublet, with another much higher band, here. Here is another FOX3 monoclonal from ThermoFisher showing something unexpected in heart tissues and no obvious band at ~70kDa here. This antibody was made against the N-terminal sequence of FOX3 but exact details are not given. The iReal company has that ~70kDa band on CNS extracts, but not in heart, see here. Western blot data from Hellobio is confusing as the antibody was made aginst the C-terminal of FOX3 but nevertheless still shows a ~70kDa band here. There is no obvious similarity between synasin 1 and that region of FOX3. Here's another antibody from German Research Products here, made against the N-terminal peptide and showing the ~70kDa band
In conclusion current data suggests that the ~70kDa band seen by multiple antibodies is synapsin 1 which shows up on blots since it is a very high abundance protein with low sequence homology to FOX3/NeuN. In contrast FOX3/NeuN is, like most RNA binding proteins, of very low abundance and is only revealed robustly since the A60, MCA-1B7 and other antibodies are of high affinity. The FOX3/NeuN epitope we defined is close to the N-terminus and so should be accessible to antibodies while the putative synapsin I epitope is buried within the sequence. This suggests that the synapsin I epitope may be partially or wholly masked in fixed sections. In line with this suggestion, Mao et al. showed that saponin treatment of sections increased the A60 staining of hippocampal mossy fibres. Our advice is don't worry about the ~70kDa band as it has virtually no impact on cell and tissue staining. A60, MCA-1B7 and other FOX3/NeuN antibodies are very widely utilized as neuronal markers and have been since the Mullen et al paper came out in 1992. Since we are very confident in the utility and specificity of MCA-1B7 we have cloned, sequenced, expressed and purified it in a recombinant form. Many journals now prefer scientists to use recombinant antibodies if they are available, so we made the new products MRA-1B7 and RRA-1B7. The former is a recombinant form of MCA-1B7 and the later is a recombinant form in which the mouse Ig constant regions have been replaced with those of rabbit. These versions increase the utility of this widely used antibody going forward and provide insurance that these antibodies will be avaliable in perpetuity. We also note that our antibody has a much more detailed characterization than any other available reagent.
