in line mals  (Waters Corporation)

Journal: The Journal of Biological Chemistry

Article Title: Cleavage at the nsp5–nsp6 site of SARS-CoV-2 main protease intermediate precursor is faster from a monomer than a dimer form

doi: 10.1016/j.jbc.2026.111395

Figure Lengend Snippet: Time course of the processing of MPro precursors at its termini by MPro WT and characterization of the products by SDS-PAGE and SEC–MALS. A , schematic showing the precursor and products that result from cleavage by MPro WT . B and C , time course of the processing of precursors (50 μM) by MPro WT (1 μM). Samples (5 μg/lane) were analyzed by SDS-PAGE. D , SDS-PAGE of flow-through (FT) and bound (B) fractions (3 μg/lane) following nickel affinity chromatography of digest like that shown in panel ( B , 21 h). S and P refer to control lanes (2 μg/lane), Precursor C145A and mature MPro C145A , respectively. E – J , characterization of the precursor and products by SEC–MALS analyses. E , precursor C145A . F , products of the digest as shown in panel ( B , lane 21 h) of Precursor C145A (77 μM) incubated with MPro WT (1 μM). G and H , bound ( B ) and FT fractions derived from panel ( D ) corresponding to MPro C145A-IP and MBP product peaks, respectively. I , a low injection concentration of the MPro C145A-IP product. J , a low injection concentration of mature MPro C145A . D and M in parentheses refer to the predominantly dimer and monomer species, respectively, at the estimated concentration indicated below the molecular mass. M and kDa denote molecular weight markers and kilodaltons, respectively. Black circles indicate the molecular mass across the peak slice. IP, intermediate precursor; MPro, main protease; MPro WT , mature WT MPro; SEC–MALS, size-exclusion chromatography with multiangle light scattering.

Article Snippet: Proteins were fractionated by analytical SEC with in-line MALS (DAWN Heleos-II; Wyatt Technology, Inc), refractive index (Optilab T-Rex; Wyatt Technology, Inc), and UV (Waters 2487; Waters Corporation) detectors.

Techniques: SDS Page, Affinity Chromatography, Control, Incubation, Derivative Assay, Injection, Concentration Assay, Molecular Weight, Size-exclusion Chromatography, Multi-Angle Light Scattering

Journal: The Journal of Biological Chemistry

Article Title: Cleavage at the nsp5–nsp6 site of SARS-CoV-2 main protease intermediate precursor is faster from a monomer than a dimer form

doi: 10.1016/j.jbc.2026.111395

Figure Lengend Snippet: Comparison of monomeric MPro in the absence and presence of C-terminal (+3) -GB1-6H (IP) residues by DSF and SV-AUC, respectively. A , monomer–dimer distribution of MPro M in the absence and presence of C-terminal (+3) -GB1-6H (IP) residues. Red trace in ( A ) shows a comparison of MPro C145A-IP in the absence of M mutations at the lowest concentration tested (0.34 μM at 230 nm) being consistent with the SEC–MALS result ( I ). Samples were analyzed in 3 mm pathlength cells, except for MPro C145A-IP in 12 mm cells. M and D denote monomer and dimer, respectively. B , DSF profiles of mature MPro constructs. Δ T m defines the difference in T m in the absence and presence of mutations M (E290A/R298A) and C145A. DSF, differential scanning fluorimetry; IP, intermediate precursor; MPro, main protease; SEC–MALS, size-exclusion chromatography with multiangle light scattering; SV-AUC, sedimentation velocity analytical ultracentrifugation.

Article Snippet: Proteins were fractionated by analytical SEC with in-line MALS (DAWN Heleos-II; Wyatt Technology, Inc), refractive index (Optilab T-Rex; Wyatt Technology, Inc), and UV (Waters 2487; Waters Corporation) detectors.

Techniques: Comparison, Concentration Assay, Construct, Size-exclusion Chromatography, Multi-Angle Light Scattering, Sedimentation, Analytical Ultracentrifugation