chk2 enzyme solution  (Carna Inc)

Journal: Frontiers in Immunology

Article Title: SARS-CoV-2 Antibodies Mediate Complement and Cellular Driven Inflammation

doi: 10.3389/fimmu.2021.767981

Figure Lengend Snippet: Complement deposition in recovered SARS-CoV-2 individuals. (A) Measurements of complement components C4, C3 and TCC in convalescent EDTA plasma. Groups divided according to levels of total IgG; high, intermediate, low and negative healthy controls, n = 240. Levels were assessed by coating plates with 3 µg/nm RBD, followed by incubation with EDTA plasma diluted 1:60 (for high IgG samples) and 1:20 (for intermediate, low and negative IgG samples). A normal human serum pool was applied in a 1:50 dilution. Biotinylated monoclonal antibodies against C4b, C3bc and TCC (2 µg/ml) was used for detection. Dynamic range represented in arbitrary units (AU). A p value < 0.05 was considered significant. ***p < 0.001 using Tukey all-pair comparisons. (B) The correlation between deposition of either C4, C3 or TCC and IgG levels using Spearman rank correlation analysis.

Article Snippet: Biotinylated monoclonal antibodies against C4b (Hyb 162-02, Bioporto Diagnostics), C3bc [in-house produced, clone BH6 ( )] and TCC [in-house produced, clone aE11 ( )] were applied in a concentration of 2 μg/ml in PBS-T and incubated for 1 h RT.

Techniques: Clinical Proteomics, Incubation, Bioprocessing

Journal: bioRxiv

Article Title: Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

doi: 10.1101/2021.09.23.461563

Figure Lengend Snippet: A) Enzymatic cleavage by C1s of the small peptide substrate Z-L-Lys-sBzl was assayed with DTNB (Ellman’s reagent) in the presence of 25 μM BBK32-C (non-inhibitory control) or ErpQ at 25°C for 1hr. Experiments were performed in triplicate. Absorbance was read at 412 nm and signals were normalized to negative control no-substrate wells. B) Top: Proteolytic cleavage of C2 by C1s enzyme produces ∼70kDa C2b and ∼35kDa C2a after 1hr at 37°C. Lanes 1-5: C2b accumulation in the presence (“+”) or absence (“-“) or 25 µM ErpQ, 25 µ M BBK32-C (non-inhibitory control), 6.25 nM C1s, and 685 nM C2. (Note that the amount of C1s loaded is below the level of detection by SDS-PAGE). Lanes 6-13: C2b accumulation in the presence of 6.25 nM C1s, 685 nM C2 and a two-fold dilution series (from 16 to 0.13 μM) of ErpQ. Bottom: The fraction of C2b relative to total input C2 in the same lane determined by densitometry analysis data are normalized to C2 (lane 5) and C1s digested C2 (lane 6). A representative gel is shown. The experiment was performed three times. C) Top: C4, which consists of 3 polypeptide chains, C4α (97 kDa), C4β (77 kDa), C4γ (33 kDa), is cleaved by C1s enzyme for 1hr at 37 °C to produce C4α’ (88 kDa). Lanes 1-5: SDS-PAGE profile in the presence (“+”) or absence (“-“) or 25 µ M ErpQ, 25 µ M BBK32-C (non-inhibitory control), 6.25 nM C1s, and 616 nM C4. Lanes 6-13: SDS-PAGE profile in the presence of 6.25 nM C1s, 616 nM C4 and a two-fold dilution series (from 25 to 0.20 μM) of ErpQ. Bottom: The fraction of C4α’ relative to input C4β in the same lane and normalized to C1s + C4 positive control (lane 6) and negative control C4 (lane 5) was determined by densitometry analysis.

Article Snippet: A 1:300 dilution anti-C4 antibody (HYB 162-02) (Santa Cruz Biotechnology) in CP Buffer incubated at 37°C for 1 hour was used to detect complement activation.

Techniques: Control, Negative Control, SDS Page, Positive Control