The name is absent

17

human, rabbit and cat, and that it should not cross react with other known proteins. The
anti-HCN3 antibody’s epitope was amino acids 625-774 near the С-terminus of the human
HCN3 protein.

Mouse brain and 12 salamander retinas were extracted and homogenized in a buffer con-
sisting of 500mM NaCl, 20 mM Tris-HCL buffer at pH 7.5 (Bio-Rad), 2mM EGTA (Fluka)
and a protease inhibitor cocktail tablet (Roche). Extracts were kept on ice, and centrifuged
at 4 C at 500 g for 20 minutes. The supernatant was extracted and pellet discarded. For the
mouse brain, in order to isolate membrane proteins, the extract was centrifuged at 30000
g for 20 minutes at 4 C and the pellet was collected. Runs of non-ultracentrifuged brain
extract gave identical results to the ultracentrifuged extract. Ultracentrifuged retina did not
yield enough protein pellet to stain. Ultracentrifuged brain was resuspended in 10% SDS.
Extracts were combined 1:1 into a Lamaelli buffer (Bio-Rad) and denatured at 80 C for 10
minutes. Solutions were run in a 7.5% Tris Ready-gel (Bio-Rad) for 45 minutes at 100 V,
and transferred for 60 min at 110 V to a PVDF membrane pre-soaked in methanol. Transfer
buffer was TrisZGlycine + 20% methanol.

The membranes were incubated in antibodies at a 1:200 dilution in 3% milk overnight.
After three sets of washes, they were incubated once again with HRP conjugated anti-rabbit
antibody at a 1:500 dilution. Membranes were washed 3x again, and reacted with ECP de-
tection solution. The reaction was exposed onto X-ray film for either 5 or 10 minutes and
developed. BLASTp searches on antibody epitope were performed on Xenopus tropicalis
data obtained from Xenbase.org [20].

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