Fig. 4.

Biotinylated protein pull-down. (A) Blood, plasma, serum and PEG-plasma were incubated with (+) or without (−) biotin–PEG₄–hydrazide probe for 1 h at 52 °C. Biotinylated proteins were captured by SA-resin, eluted by boiling the resin in SDS loading buffer and visualised by 6% SDS-PAGE and Coomassie staining. (B) Western blot of biotin–PEG₄–hydrazide treated blood, plasma, serum and PEG-plasma pre (−) and post (+) SA capture. Biotinylated proteins detected by SA-HRP. (C) Total C3 protein in reaction mix pre (−) and post (+) SA capture. Two known amounts of C3(bio) were treated with SA-resin and the non-captured remaining protein detected by SDS-PAGE, with lumeitin stain. (D) Table of identified peptides from pull-down experiment (A). Gel bands i–v were excised, and mass fingerprint established by tryptic digest and MALDI-TOF MS, searched against the Swissprot database by the Mascot search engine. All identified proteins (with scores greater than 56 and i.e., significant (p < 0.05)) are listed with ID, score, number of matched peptides and % sequence coverage. (E) Sequence coverage of the identified C3 protein from excision v. β-chain shown in italics, identified peptides highlighted in grey, the GCGEQ (the internal thioester) is boxed, and the Ana (C3a) domain underlined (cleaved during C3 activation by C3 convertase).