and D

and D.A.K. been shown that crows and cockatoos can develop tools to solve complex jobs2C4, pigeons can memorize abstract visual patterns5,6, songbirds Eprodisate are able to learn conspecific and novel vocalizations7,8, Eprodisate and robins rely on magnetic fields for long-range navigation9,10. It is apparent from these studies the avian brain processes information from a myriad of sensory cues allowing it to drive an array of complex behaviours. The quantitation of immediate early gene (IEG) manifestation is a well established strategy to map the neuronal networks that integrate this information. IEGs symbolize a class of genes characterized by a rapid rise in manifestation levels upon sustained cell stimulation and are therefore linked to synaptic activity in the mind11,12. One popular IEG is the transcription element ZENK (an acronym for hybridization, or a commercial polyclonal antibody raised against the rabbit ZENK homologue EGR-1 (C-19)19,27C30. While C-19 has been widely used, it displays high batch-to-batch variance, requires laborious amplification methods in some varieties27,28, and its production has now been discontinued from the supplier. In order to establish a reliable alternative to C-19 we set out to generate a ZENK antibody specifically for (clZENK) we extracted mRNA from your pigeon brain, generated cDNA, and amplified the transcript using primers designed from existing genomic resources31,32. This resulted in the cloning of a 1443?bp gene product that codes for any 481 amino acid protein. An N-terminal Pdgfra fragment 260 amino acids in length (1C260) with high surface probability was chosen as the antigen. The protein was heterologously indicated in E. coli, purified, the sequence verified by mass spectrometry, and then injected into Eprodisate BALB/c mice. The spleens of immunized mice were harvested, hybridomas generated, and clones screened by western blot analysis for sera that bind the clZENK protein (Fig.?1a). This resulted in the recognition of clone 7B7-A3. Purified antibodies generated from this clone detect ZENK (~70?kDa), and GFP tagged ZENK (~95?kDa) in protein lysates from pigeon embryonic fibroblasts (PEFs) transiently transfected with clZENK-GFP (Fig.?1b). In lysates generated from your pigeon mind we observed a single 70?kDa band that is consistent with endogenously expressed ZENK. It should be mentioned that like some commercially available antibodies raised against ZENK the recognized size of 70?kDa in european blots exceeds the predicted excess weight of 57?kDa, possibly due to posttranslational modifications. Consequently to validate the selectivity for the clZENK epitope, we performed a peptide competition experiment by preincubating the antibody with the antigen. This preincubation prevents binding of the 7B7-A3 antibody (Fig.?1c), but not binding of a GFP antibody control (Fig.?1d). Next, we assessed the utility of the 7B7-A3 antibody like a marker on histological sections. Utilizing antigen retrieval and a long term staining protocol we found that 7B7-A3 antibody reliably labels cell nuclei in pigeon mind sections at dilutions as low as 1:500,000 (3.2?ng/ml), without the need for post-chromogenic enhancement (Fig.?1e,f). Pre-absorption of the 7B7-A3 antibody with the antigen (Fig.?1g,h), as well as omission of the primary antibody (Fig.?1i,j) resulted in no visible nuclear staining, confirming the selectivity of the 7B7-A3 antibody for the clZENK epitope. Taken collectively, these data show the 7B7-A3 antibody binds clZENK. Open in a separate windowpane Number 1 ZENK antibody generation and validation. (a) Diagram depicting the strategy employed to generate the ZENK antibody. A 260 amino acid N-terminal fragment (purple) of the pigeon ZENK protein (orange) was recombinantly indicated and injected into mice. Harvested spleen cells were fused with myeloma cells and hybridomas screened by western blot analysis. This resulted in the recognition of clone 7B7-A3 (demonstrated in purple). (b) Western blot analysis of pigeon embryonic fibroblasts expressing ZENK-GFP or GFP only, and protein lysates from pigeon mind cells. Blots incubated with 7B7-A3 (remaining panel) show bands at ~70?kDa and ~95?kDa, corresponding to the endogenous and GFP tagged protein, respectively. (c) and (d) Peptide competition experiment. Preincubation of the antibody with the antigen (+) prevents binding of.