acrylamide gradient sds page gels  (Bio-Rad)

Journal: Cell reports

Article Title: Role of the afferent lymph as an immunological conduit to analyze tissue antigenic and inflammatory load

doi: 10.1016/j.celrep.2024.114311

Figure Lengend Snippet: (A) Pre-nodal mesenteric lymphatic vessel draining to the mesenteric node and inset of the same pre-nodal lymphatic vessel following peri-lymphatic fat removal. (B) Pipette tip (2–4 μm) showing the collected lymph fluid from the pre-nodal lymphatic vessel. (C) Pre-nodal cervical afferent lymphatic vessel draining to the deep cervical node. (D) Detail of the cannulation of a pre-nodal lymphatic vessel. (E) Separation of the mesenteric and cervical lymph proteome (3 μg of protein) on a silver-stained 4%–20% gradient acrylamide SDS-PAGE. (F) Deep Venn area proportional diagram displaying the degree of overlap and differential expression profiling of proteins identified in the mesenteric and cervical lymph using a combination of LFQ proteomics platforms (see protocol). (G) Volcano plot depicting the significant differential expression ( n = 4, p < 0.05 by t test) of 2,752 proteins. Highlighted in blue and red are the 681 proteins showing at least 2-fold downregulation and 799 proteins showing at least 2-fold upregulation in the cervical vs. mesenteric lymph proteomes. (H) Principal component analysis (PCA), generated by MetaboAnalyst, based on the DIA intensities of the proteins identified in n = 4 biological replicates of afferent mesenteric and cervical lymph. (I–K) LFQ comparative analysis of selected proteins displaying significant differences in their relative abundance (extracted from DIA intensities) as determined in (G). Blue and orange dots depict the mesenteric and cervical lymph proteins respectively. Statistical significance was determined using the Holm-Sidak method, with alpha = 0.05: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (L) Euclidian Ward’s dual heatmap of the top 2,000 proteins identified in all four biological replicates of mouse pre-nodal mesenteric and cervical lymph. The map highlights the significant difference in the proteomic signature of the two biological fluids harvested from the distinct anatomical regions. Only proteins that passed a selected significance statistical threshold (ANOVA/t test applied in PEAKS XPro, p < 0.05) are displayed in the heatmap. (M and N) The subsequently generated METASCAPE analysis of protein networks and GO annotations defining the different molecular functions and metabolic pathways of proteins from mesenteric (M) vs. cervical (N) lymph. All proteins and details of LFQ analysis and GO annotations related to the mesenteric and cervical proteomic analysis are presented in .

Article Snippet: Lymph proteins (10–20 μg) were mixed with the sample buffer, heated at 95 °C for 5 min, and run on a 4–20% gradient acrylamide SDS (PAGE) gels (Novex, Tris-Glycine Mini Gels, cat# XV04205PK20, from ThermoFisher Scientific) at 160 V constant following the manufacturer protocols.

Techniques: Transferring, Staining, SDS Page, Quantitative Proteomics, Generated

Journal: Cell reports

Article Title: Role of the afferent lymph as an immunological conduit to analyze tissue antigenic and inflammatory load

doi: 10.1016/j.celrep.2024.114311

Figure Lengend Snippet:

Article Snippet: Lymph proteins (10–20 μg) were mixed with the sample buffer, heated at 95 °C for 5 min, and run on a 4–20% gradient acrylamide SDS (PAGE) gels (Novex, Tris-Glycine Mini Gels, cat# XV04205PK20, from ThermoFisher Scientific) at 160 V constant following the manufacturer protocols.

Techniques: Recombinant, Protease Inhibitor, Western Blot, Bicinchoninic Acid Protein Assay, Saline, Purification, Membrane, Sterility, Control, Software, Microscopy, Mass Spectrometry