biotinylated epo mouse antibody  (R&D Systems)

Journal: Scientific reports

Article Title: EV-3, an endogenous human erythropoietin isoform with distinct functional relevance.

doi: 10.1038/s41598-017-03167-0

Figure Lengend Snippet: Figure 4. Isolation of hEPOΔ3 from HepG2 cells and detection of hEPOΔ3 derived protein EV-3 and EPO in HepG2 cell culture supernatant. (a) The nested PCR approach amplifies EPO full-length and hEPOΔ3 transcripts from HepG2 cells. (b) Captured proteins of HepG2 supernatants (α-EPO IP, using biotinylated anti-EPO antibody BAF959) are visualized by western blotting using a polyclonal rhEPO-antibody (H-162). The Western Blot shows two EPO-antibody reactive proteins (prot. 1 and prot. 2) running at the same size of recombinantly produced EV-3 (rEV-3) and EPO (two different batches of rEPO). (c) Densitometry analysis was done on the Western Blot image (jpeg) shown in Fig. 4b. The plot profile of the HepG2 immunoprecipitate in lane 5 (ROI3) reveals a faint but distinct second peak, corresponding to the plot profile of EV-3 loaded in lane 1 (ROI1). (d) EV-3, indicated by the specific peptide ‘DITVGQQAVE’, was detected in HepG2 supernatants by nano-LC-ESI-MS/MS. Quantification of EV-3 specific peptide (fmol/µg total protein of analysed sample) in sample H1, the immunoprecipitate (α-EPO-IP) of HepG2 supernatant, or sample H2, HepG2 supernatant enriched for glycoproteins by WGA chromatography.

Article Snippet: For immunoprecipitation, biotinylated anti-EPO antibody (0.5 μg; BAF959, R&D) was added to concentrated supernatant (750 μl) and incubated over-night at 4 °C on a rotating shaker.

Techniques: Isolation, Derivative Assay, Cell Culture, Nested PCR, Western Blot, Produced, Tandem Mass Spectroscopy, Chromatography