immunosorbent assay elisa kit  (Novus Biologicals)

Journal: The Journal of Cell Biology

Article Title: Assembly and phosphoregulatory mechanisms of the budding yeast outer kinetochore KMN complex

doi: 10.1083/jcb.202506015

Figure Lengend Snippet: Regulation of Mis12c Mtw1c auto-inhibition by Aurora B kinase and its effect on the interaction with CENP-C Mif2 . (A) Structural alignment of the crystal structure of K. lactis CENP-C Mif2 residues 4–36 ( Kl CENP-C Mif2_4-36 ) as bound to the Kl Mis12c Mtw1c head 1 domain (PDB: 5T59 ) to the experimental model of Sc Mis12c Mtw1c (this study). The models are colored by chain; Mis12 Mtw1 and Nnf1 are colored identically in 5T59 and our experimentally determined model. Kl CENP-C Mif2_4-36 is shown in space filling representation to highlight lack of steric clash, and overlapping binding site, with Dsn1 AI . Only Kl CENP-C Mif2_4-36 is shown. (B) SEC elution chromatograms of attempts to reconstitute the interaction between Knl1c:Mis12c Mtw1c complexes containing different Dsn1 N-terminal truncation and/or phosphomimetic mutants and CENP-C Mif2 residues 1–63 (CENP-C Mif2_1-63 ) indicated within red boxes. (C) Coomassie brilliant blue–stained SDS-PAGE gels of the chromatograms shown in B. (B and C) K HB-RWD M = full-length Dsn1. K HB-RWD M 2E = full-length Dsn1 containing Dsn1 S240E and Dsn1 S250E phosphomimetic mutations. K HB-RWD M Dsn1Δ257 = Dsn1 residues 1–257 are deleted (Dsn1 Δ1-257 ). K HB-RWD M Dsn1Δ226 = Dsn1 residues 1–226 are deleted (Dsn1 Δ1-226 ). K HB-RWD M Dsn1Δ226-2E = Dsn1 residues 1–226 are deleted and Dsn1 S240E and Dsn1 S250E phosphomimetic mutations (Dsn1 Δ1-226,S240E,S250E ). CENP-C Mif2_1-63 is indicated within a red box. (D) ITC data for (i) CENP-C Mif2_1-38 interactions with KM, and (ii) CENP-C Mif2_1-38 interactions with KM 2E . K D and n values are an average from three experiments (technical repeats). Source data are available for this figure: .

Article Snippet: CENP-U Ame1_1-33 (MDRDTKLAFRLRGSHSRRTDDIDDDVIVFKTPNW-amide), CENP-U Ame1_1-33-Mut (MDRDTKLAFRLRGSHSRRTDDIDDAAAVAKAPNW-amide), and CENP-C Mif2_1–38 (MDYMKLGLKSRKTGIDVKQDIPKDEYSMENIDDFFKDD-amide) peptides were synthesized by Alta BioScience.

Techniques: Inhibition, Binding Assay, Staining, SDS Page

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Secretion of therapeutic proteins with a-BLAST and d-BLAST. (a) The human preproinsulin (preproINS) construct contains a signal peptide, B-chain (yellow), C-peptide (dark gray), and A-chain (yellow), with engineered Furin cleavage sites flanking the C-peptide for maturation. To facilitate efficient processing within the Golgi apparatus, Furin protease was co-transfected with the BLAST modules. (b) Kinetic profiling of light-induced insulin secretion. Summary graphs of secreted C-peptide levels, quantified by ELISA, as a proxy for insulin secretion from a-BLAST (left) and d-BLAST (right). Both systems exhibited significant, time-dependent insulin release starting from 2 h of illumination (8.2-fold for a-BLAST, 8.4-fold for d-BLAST), reaching maximal induction at 24 h (13.8-fold for a-BLAST, 19.3-fold for d-BLAST). (c) Plasmid configurations for IL-12 secretion. Schematic of the heterodimeric cytokine IL-12-a-BLAST (left) and d-BLAST-IL-12 (right) constructs. (d) Kinetic profiling of light-induced IL-12 secretion. Summary graphs showing IL-12 secretion levels measured by ELISA. Significant secretion was observed starting at 3 h for a-BLAST (2.5-fold) and 2 h for d-BLAST (2.2-fold). At the 24 h time point, d-BLAST (4.7-fold) demonstrated a slightly higher dynamic range compared to a-BLAST (4.5-fold). Open circles represent individual measurements from three biologically independent samples. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Secreted human C-peptide levels in the supernatant were quantified using a Human C-peptide ELISA Kit (R&D Systems; Catalog #DICP00).

Techniques: Construct, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Secretion of therapeutic proteins with a-BLAST and d-BLAST. (a) The human preproinsulin (preproINS) construct contains a signal peptide, B-chain (yellow), C-peptide (dark gray), and A-chain (yellow), with engineered Furin cleavage sites flanking the C-peptide for maturation. To facilitate efficient processing within the Golgi apparatus, Furin protease was co-transfected with the BLAST modules. (b) Kinetic profiling of light-induced insulin secretion. Summary graphs of secreted C-peptide levels, quantified by ELISA, as a proxy for insulin secretion from a-BLAST (left) and d-BLAST (right). Both systems exhibited significant, time-dependent insulin release starting from 2 h of illumination (8.2-fold for a-BLAST, 8.4-fold for d-BLAST), reaching maximal induction at 24 h (13.8-fold for a-BLAST, 19.3-fold for d-BLAST). (c) Plasmid configurations for IL-12 secretion. Schematic of the heterodimeric cytokine IL-12-a-BLAST (left) and d-BLAST-IL-12 (right) constructs. (d) Kinetic profiling of light-induced IL-12 secretion. Summary graphs showing IL-12 secretion levels measured by ELISA. Significant secretion was observed starting at 3 h for a-BLAST (2.5-fold) and 2 h for d-BLAST (2.2-fold). At the 24 h time point, d-BLAST (4.7-fold) demonstrated a slightly higher dynamic range compared to a-BLAST (4.5-fold). Open circles represent individual measurements from three biologically independent samples. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Secreted human C-peptide levels in the supernatant were quantified using a Human C-peptide ELISA Kit (R&D Systems; Catalog #DICP00).

Techniques: Construct, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation