blue native page  (Bio-Rad)

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

Journal: CNS Neuroscience & Therapeutics

Article Title: NOTCH3 Mutation Causes Glymphatic Impairment and Promotes Brain Senescence in CADASIL

doi: 10.1111/cns.70140

Figure Lengend Snippet: Diminished AQP4 expression in NOTCH3‐R170C astrocyte. (A–C) Brain cells of NOTCH3‐R170C and ‐WT mice (16 weeks of age) were subjected to single cell RNA sequencing (scRNAseq). (A) Left: Merged Uniform manifold approximation and projection (UMAP) plot representing 15 color‐coded cell clusters identified in the combined single‐cell transcriptomes. Cluster names were manually assigned. Right: Feature plots of Notch3 . (B) Expression of Notch3 among brain cells. (C) Violin plots of Notch3 and Aqp4 expression in astrocytes. (D) Brain protein of NOTCH3‐R170C or ‐WT mice (24 weeks of age) was subjected to western blot to evaluate AQP4 expression. N = 3 in each group. ** p < 0.01; by Student's t test (mean ± SEM). (E, F) Primary NOTCH3‐R170C and ‐WT astrocyte cultures were prepared. (E) Aqp4 mRNA level was evaluated with RT‐PCR. (F) AQP4 protein expression in whole cell and plasma membrane of astrocytes was assessed with western blot. Experiments were repeated for three times. * p < 0.05; by Student's t test. Difference of means (± SEM) is displayed. (G) Primary NOTCH3‐R170C and ‐WT astrocyte cultures were subjected to BN page and then 2D SDS page to evaluate the orthogonal arrays of particles (OAPs) of AQP4. (H) Coronal brain sections of NOTCH3‐R170C or ‐WT mice (24 weeks of age) were subjected to immunostaining of GFAP (green) and AQP4 (red). The AQP4 polarization was calculated by AQP4 perivascular endfeet area. White arrowheads indicated the depolarized AQP4 distribution. N = 6 in NOTCH3‐WT group and N = 5 in NOTCH3‐R170C group. * p < 0.05; by Student's t test (mean ± SEM).

Article Snippet: The protein content of the supernatant was diluted by 2X Blue Native PAGE Sample Buffer (Sangon Biotech, C506055‐0005).

Techniques: Expressing, RNA Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Clinical Proteomics, Membrane, SDS Page, Immunostaining