Journal: Molecular & Cellular Proteomics

Article Title: A Tandem Affinity Purification-based Technology Platform to Study the Cell Cycle Interactome in Arabidopsis thaliana

doi: 10.1074/mcp.m700078-mcp200

Figure Lengend Snippet: FIG. 4. Protein complex analysis by BN/SDS-PAGE (A) and size exclusion chromatography (B). A, protein complexes fractionated in the first dimension by BN-PAGE. In the second dimension, proteins were separated by SDS-PAGE, and gels were immunoblotted with the anti-CDKA;1 antiserum (1:5000). For CDKA;135S, 400 g of protein was analyzed from the total protein extract (panel 1) and the IgG-unbound fraction (panel 2), and 55 g was analyzed from the final calmodulin eluate (panel 3). Recombinant CDKA;1 and CDKA;1 fused to the complete TAP tag and the CBP tag are indicated as CDKA;1-TAP and CDKA;1-CBP, respectively. B, CDKA;1-TAP eluate (2 ml) fractionated on a Superdex 200 (300/10) size exclusion chromatography column. From each 500-l fraction, 25 l was assayed for kinase activity with histone H1 as a substrate (panel 1), and 10 l was analyzed for the presence of the bait protein by immunoblotting with an antiserum against CDKA;1 (1:5000) (panel 2). Arrowheads indicate the elution positions of marker proteins with their molecular masses (kDa). Std, standard.

Article Snippet: Briefly total protein extract was incubated for 1 h at 4 °C under gentle rotation with 500 l of IgG-Sepharose 6 Fast Flow beads (GE Healthcare) pre-equilibrated with 10 ml of extraction buffer.

Techniques: SDS Page, Size-exclusion Chromatography, Recombinant, Activity Assay, Western Blot, Marker

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The lectin complement pathway serine proteases (MASPs) represent a possible crossroad between the coagulation and complement systems in thromboinflammation.

doi: 10.1111/jth.13208

Figure Lengend Snippet: Fig. 4. Panels A–E: binding of LP components from serially diluted lepirudin-PPP (starting with 3% dilution) to plasmin-generated fragments of fibrinogen and fibrin coating microtiter plates. Detection was carried out using specific biotinylated mAbs followed by streptavidin-HRP (n = 3). Panel A: negligible binding of MBL, Ficolin-1, Ficolin-2 and Ficolin-3 to fibrin fragment DD. Panel B: significant binding of MASP-1 and, to a lesser degree, MASP-2 to fibrin fragment DD. Insignificant binding of MASP-1 and MASP-2 to fibrin fragment E (panel C), fibrino- gen fragment D (panel D) and fibrinogen fragment E (panel E). Panels F–G: generation of MASP-1 and MASP-2/AT complexes by fibrin and fibrin fragment DD. Lepirudin-PPP (n = 3) was incubated with increasing concentrations of fibrin (0–12 lg mL1, panel F) or fibrin fragment DD (0–25 lg mL1, panel G). After incubation MASP-1 and MASP-2/AT/C1-INH complexes were quantified by sandwich ELISA. In PPP in contact with fibrin, MASP-1/AT and MASP-2/AT complexes were preferentially formed (panel F). Fibrin fragment DD induced similar genera- tion of both MASP-1, MASP-2/AT and MASP-1 and MASP-2/C1-INH, but the latter were formed at a much lower concentration of DD (panel G). LP, lectin pathway; PPP, platelet-poor plasma; MBL, mannose-binding lectin; AT, antithrombin.

Article Snippet: 4 The wells were then incubated with 60 lL of HRP-conjugated streptavidin (GE Healthcare) diluted 1 : 500.

Techniques: Binding Assay, Generated, Incubation, Sandwich ELISA, Concentration Assay, Clinical Proteomics