blue native page analysis  (Bio-Rad)

Journal: Molecular cell

Article Title: Structure and functionality of a multimeric human COQ7:COQ9 complex.

doi: 10.1016/j.molcel.2022.10.003

Figure Lengend Snippet: Figure 1. Structure-function characterization of COQ7 (A) Native PAGE analysis of purified GB1-Nd38COQ7:His6-Nd79COQ9 complex. (B) Structure of COQ7 with visible channel leading to an active site. Iron atoms were not resolved by cryo-EM and were added based on the structure of bac- toferritin (PDB: 4AM2). (C) Electrostatic surface potential of COQ7 with visible entry to the hydrophobic channel. (D) The hydrophobic channel with visible additional ligand density inside. Important residues are labeled. Corresponding residues in yeast are in parentheses. (E and F) Octaprenylphenol (OPP) and coenzyme Q8 (CoQ8) in E. coli cells and the GB1-Nd38COQ7:His6-Nd79COQ9 complex purified from E. coli (mean ± SD, n = 2). (G) Growth of Coq7 mutants. Cells start with fermentation and then switch to respiration (mean ± SD, n = 3). (*) corresponds to time point used for measurements in (H)–(J). (H) Coenzyme Q6 level in Coq7 mutants (mean ± SD; n = 3; one-sided Student’s t test; *p < 0.05; n.s., not significant). (I) Expression level of the Coq7 yeast mutants. Native Coq7 in BY4742 strain is below the limit of detection. (J) DMQ6 level in Coq7 mutants. (mean ± SD; n = 3; one-sided Student’s t test; *p < 0.05; n.s., not significant).

Article Snippet: Supernatants were combined, concentrated to 1mL using Amicon Centrifugal Filter (15k Da cut-off) and subjected to Size Exclusion Chromatography and subsequent blue native PAGE analysis (NativePAGE, 4-16%, Bis-Tris, Invitrogen).

Techniques: Clear Native PAGE, Cryo-EM Sample Prep, Labeling, Expressing

Journal: Molecular cell

Article Title: Structure and functionality of a multimeric human COQ7:COQ9 complex.

doi: 10.1016/j.molcel.2022.10.003

Figure Lengend Snippet: Figure 3. Structure of COQ7:COQ9 heterodimer in lipid environment (A) Snapshot of the COQ7:COQ9 heterodimer structure with visible lipids (light gray), NADH (green), and OPP (yellow). (B) Details of the COQ7:COQ9 interaction interface. (C) Native PAGE of the GB1-Nd38COQ7:His6-Nd79COQ9W240K complex. (D) Zoom on lipid molecules forming a lipid pseudo-bilayer. (E) Zoom on the tilted lipid head groups (orange and red). (F) Zoom on lipid-binding substrate pocket in COQ9 surrounded by surface lipids. No quinone density is detected around the substrate-binding COQ9’s W240 residue (magenta). (G) LC-MS/MS lipidomic analysis of OPP content in individually purified His6-GB1-Nd38COQ7, His6-Nd79COQ9, or the full GB1-Nd38COQ7:His6-Nd79COQ9 complex (mean ± SD, n = 2).

Article Snippet: Supernatants were combined, concentrated to 1mL using Amicon Centrifugal Filter (15k Da cut-off) and subjected to Size Exclusion Chromatography and subsequent blue native PAGE analysis (NativePAGE, 4-16%, Bis-Tris, Invitrogen).

Techniques: Clear Native PAGE, Binding Assay, Residue, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular cell

Article Title: Structure and functionality of a multimeric human COQ7:COQ9 complex.

doi: 10.1016/j.molcel.2022.10.003

Figure Lengend Snippet: Figure 6. Two COQ7:COQ9 tetramers form an octamer (A) Structure of the octamer with visible bound lipids (white), NADH (green), and OPP (yellow). (B) Hydrophobic interior of the octamer displayed as electrostatic surface of one of the COQ7:COQ9 tetramers. Stabilizing lipids (white), NADH (green), and OPP (yellow) shown. (C) SDS-PAGE analysis of protein stability upon delipidation of the GB1-Nd38COQ7:His6-Nd79COQ9 with BioBeads; T0—soluble fraction before delipidation, T60 BioBeads—protein bound to beads after 60-m incubation, T60 delipidated—soluble fraction after 60-m delipidation, and T60 not delipidated—soluble fraction after 60-m incubation without BioBeads. (D) Native PAGE analysis of the GB1-Nd38COQ7:His6-Nd79COQ9 complex after 1-h delipidation. (E) DSF analysis of melting temperatures of individually purified His6-Nd38COQ7, His6-Nd79COQ9, the mixture of the two proteins, and the GB1-Nd38COQ7:- His6-Nd79COQ9 complex.

Article Snippet: Supernatants were combined, concentrated to 1mL using Amicon Centrifugal Filter (15k Da cut-off) and subjected to Size Exclusion Chromatography and subsequent blue native PAGE analysis (NativePAGE, 4-16%, Bis-Tris, Invitrogen).

Techniques: SDS Page, Incubation, Clear Native PAGE

Journal: Human Vaccines & Immunotherapeutics

Article Title: Pentameric M2e influenza vaccine candidate generates strong immunity with limited survival benefit

doi: 10.1080/21645515.2025.2610073

Figure Lengend Snippet: Designing and expression of soluble M2e-5x protein in mammalian (Expi293F) cells. (A) Schematic illustration of expression cassettes used in mammalian vectors (pcDNA3.1(1)) for protein production. (B) The elution profile of M2e-5x protein on a Superdex 200, 16/60 column shows a single well-defined peak at ~11 mL corresponding to oligomeric soluble fractions. (C) (i)12% reducing SDS-PAGE image of a purified M2e-5x protein; lane 1 contains a pre-stained ladder and lane 2 contains 10 µg of M2e-5x. (ii) Western blot image of M2e-5x protein probed with anti-M2e (14C2) primary antibody and developed with anti-mouse IgG-HRP conjugated secondary antibody using femtolucent substrate. (D) Native-PAGE profile of the purified M2e-5x protein; lane 1, molecular mass marker, and lane 2, M2e-5x. (E) The long-term stability of M2e-5x protein is assessed at different time intervals by determining ELISA-based variations in binding efficiency with anti-M2e (14C2) antibody. (i) M2e-5x storage at 4°C for 10 days, 20 days, and up to 60 days. (ii) Storage of M2e-5x at 37°C for 24 hours, 48 hours, and 72 hours.

Article Snippet: The purity and oligomeric properties of the purified proteins were confirmed on SDS-PAGE and Blue native-PAGE (Mini-PROTEAN TGXTM, Bio-Rad)., For Blue native-PAGE analysis using 4% to 15% Native-PAGE gels (Mini-PROTEAN TGXTM, Bio-Rad, Hercules, CA, USA), Native-PAGE sample preparation buffer (Invitrogen, Waltham, MA, USA), and Bis-Tris running buffer were used., Proteins were transferred from SDS-PAGE to a PVDF membrane for western blotting with primary antibodies (mouse polyclonal sera, 1:500; anti-M2e 14C2, 1:1000, Abcam), and HRP-conjugated anti-mouse secondary antibody (1:2000, Jackson) was used for detection with chemiluminescence reagents (luminol and peroxidases, G Biosciences)., ,

Techniques: Expressing, SDS Page, Purification, Staining, Western Blot, Clear Native PAGE, Marker, Enzyme-linked Immunosorbent Assay, Binding Assay