Santa Cruz sc-18810
HFE protein was discovered in 1997; however, there is up to now no reliable published information describing its in vivo detection. A possible reason is the fact that the Hfe gene is expressed at very low levels – in the liver the Hfe mRNA requires almost 30 PCR cycles to show on real-time PCR.
We were therefore quite happy to discover an antibody which can detect HFE in mice and rats: The Santa Cruz polyclonal sc-18810. And we were quite sad when Santa Cruz stopped producing its polyclonals in 2016. Now our stock of the antibody is slowly running out, with no replacement is in sight.
The trick, when detecting HFE, is to ISOLATE THE MEMRANE FRACTION. In the past, we used microsomes, more recently, we have switched to the 3000 g plasma membrane-enriched fraction (see Frýdlová J et al., Effect of Erythropoietin, Iron Deficiency and Iron Overload on Liver Matriptase-2 (TMPRSS6) Protein Content in Mice and Rats. PLoS One. 2016 Feb 4;11(2):e0148540, for the procedure of 3000 g fraction preparation). The necessity of membrane enrichment is nicely illustrated in the Figure below: – in the traditional NP40 homogenate, the antibody simply does not work. Even in microsomes and plasma membranes, the detection of HFE is weak, but, at least, HFE is there to be seen! It is glycosylated (as predicted) and reacts to iron deficiency.
The antibody is relatively strong and specific: We have tested it both on Hfe knockout livers (see above) , as well as on livers overexpressing Hfe using the AAV8 system.
Santa Cruz was selling other anti HFE antibody as well, sc-18806, but in our hands, it simply did not work.