fast protein liquid chromatography fplc system  (Bio-Rad)

Journal: bioRxiv

Article Title: A major histocompatibility complex (MHC) class II molecule that binds the same viral pathogen peptide with both nonamer and decamer core sequences for presentation to T cells

doi: 10.1101/2025.10.27.684722

Figure Lengend Snippet: BL2*21:01 linked to the MDV pp38 peptide was expressed in insect cells and purified, and has a similar thermostability as other chicken class II molecules bound to MDV peptides. A. Cartoon showing full-length construct, with scissors indicating the potential cleavage sites of endoproteinase Glu-C (Endoproteinase V8). B. UV traces of FPLC size-exclusion chromatography (SEC) using Superdex S200 column, before (blue) and after (black) being subjected to Endoproteinase V8 cleavage at 37°C overnight to remove C-terminal tags and dimerization domains. C. SDS-PAGE followed by Coomassie blue staining after initial nickel column purification (Input) and SEC fractions comprising Peak1 (blue trace of profile in A). Standard protein markers with indicated molecular masses; blue arrows indicate position of α- and β-chains. D. SDS-PAGE followed by Coomassie blue staining of endoproteinase V8 on its own, Peak1 (blue trace of profile in A), and SEC fractions from Single Peak (black trace). Standard makers and blue arrows is in C, black arrows indicate the position of the endoproteinase V8 cleaved α- and β-chains. E. Thermal denaturation curves for BL2*02:01 molecules expressed with MDV peptides from glycoprotein H (gH, with melting temperature 69°C), glycoprotein E (gE, 75°C), and unique long gene 43 tegument protein (UL43, 78°C), compared to BL2*021:01 molecule expressed with MDV pp38 peptide.

Article Snippet: The His-tagged protein complex was eluted from the nickel-Sepharose with 1 M imidazole in 25 mM Tris-HCl (pH 8.5), then purified by size-exclusion chromatography on a fast protein liquid chromatography (FPLC) instrument (Amersham) using Superdex S200 in 100 mM TrisCl (pH 8.5).

Techniques: Purification, Construct, Size-exclusion Chromatography, SDS Page, Staining, Nickel Column