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. 2005 Jun 1;25(22):5455–5463. doi: 10.1523/JNEUROSCI.5123-04.2005

Figure 2.

Figure 2.

Purification of proBDNF and mature BDNF. A, B, Recombinant adenovirus encoding BDNF-His (A) or proBDNF-His (B) was used to infect HEK 293 cells, and conditioned media were purified using Ni-chromatography. Lane 1, Conditioned media before chromatography. Lane 2, Column flow-through. Lane 3, Column wash. Lane 4, Column eluate. Lane 5, Column eluate subjected to silver stain analysis. C, Purification of proBDNF-His from media of baculovirus-infected insect cells. Lane 1, Conditioned media before chromatography. Lane 2, Column flow-through. Lane 3, Column wash. Lane 4, Column eluate. Lane 5, Column eluate subjected to silver stain analysis. D, ProBDNF-His column eluates (from lane 4, B) were reprobed with antibody specific for the pro-domain of BDNF. E, Medium from 293 cells expressing proBDNF-His was harvested and purified using Ni-chromatography. Purified proteins were incubated without or with N-glycanase (25 mU/ml) at 20°C for 12 h. Reactions were terminated by addition of SDS sample buffer and boiling. Proteins were separated by SDS-PAGE and Western blotted with anti-mature BDNF. When applicable, positions of proBDNF, mature BDNF, and their deglycosylated forms are indicated by arrows. WB, Western blot.