biotin streptavidin fluorescent conjugates  (Vector Laboratories)

Journal: iScience

Article Title: Identification of skeletal muscle stem cell adhesion motifs using spot-synthesis-based peptide arrays

doi: 10.1016/j.isci.2025.114498

Figure Lengend Snippet: 2D validation of candidate peptides (A) Scheme depicts the bovine serum albumin (BSA) coating strategy for polystyrene culture dishes with subsequent peptide functionalization. After BSA activation with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC), C-terminally thiolated peptides were covalently linked to the coating substrate. (B) LAMα1-p1 peptides that were N-terminally linked to a biotin molecule were used for the functionalization of BSA coating as described in (A) and subsequently detected with fluorophore-coupled streptavidin (green). Serum free myoblast growth medium was used as a vehicle control. Scale bar = 500 μm. (C and D) Schematic workflow (C) and quantification (D) of WT myoblast numbers 7 h after plating on BSA-conjugated candidate peptides. Values are expressed relative to BSA coating without peptide functionalization (BSA). (E and F) Schematic workflow (E) and quantification of WT myoblast numbers (F) ∼2.5 days after plating on BSA-conjugated candidate peptides. Values are expressed relative to BSA coating without peptide functionalization (BSA). (G) Percentage of KI67 positive WT myoblasts 2.5 days after plating on BSA-conjugated candidate peptides. (H and I) Schematic workflow (H) and quantification of LAMA2−/− myoblast numbers (I) ∼2.5 days after plating on BSA-conjugated candidate peptides. Values are expressed relative to BSA coating without peptide functionalization (BSA). (J) Percentage of KI67 positive LAMA2−/− myoblasts 2.5 days after plating on BSA-conjugated candidate peptides. Horizontal bars represent means ± sem from n = 4 (D,I,J) and n = 3 (F,G) biological replicates, each corresponding to an independent myoblast line isolated from a separate mouse. p values relative to the unconjugated BSA control were calculated using one-way ANOVA with Dunnett correction. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After washing, the pellet was incubated with MACS streptavidin beads solution (1:200, 130-048-101, Miltenyi Biotec) for 1 h on ice.

Techniques: Biomarker Discovery, Activation Assay, Control, Isolation

Journal: iScience

Article Title: Identification of skeletal muscle stem cell adhesion motifs using spot-synthesis-based peptide arrays

doi: 10.1016/j.isci.2025.114498

Figure Lengend Snippet: In vivo peptide delivery (A) Scheme shows the maleimide chemistry conjugation strategy used to couple biotinylated LAMα1-p1 peptides to the cysteines in the agrin N-terminus (NtA-Ag) to create NtA-Ag-LAMα1-p1. (B) Western blot detects biotinylated proteins using horseradish peroxidase-coupled streptavidin with two lanes containing varying amounts of NtA-Ag, one lane of LAMα1-p1, and two lanes with varying amounts of NtA-Ag-LAMα1-p1. White arrows indicate the predicted size of NtA-Ag-LAMα1-p1. (C) Detection of biotin using fluorescent streptavidin in spots containing NtA-Ag-LAMα1-p1 or Nt-Ag on laminin 111-rich Matrigel coating. Scale bars = 500 μm. (D) Experimental design and scheme show the intramuscular injection of biotinylated NtA-Ag-LAMα1-p1. (E) Illustration shows the hypothetical presentation of candidate peptides to muscle stem cells by NtA-Ag in the skeletal muscle basal-lamina after injection. (F) Dystrophin immunostaining (dyst, green) of tibialis anterior (TA) cross sections from mice injected with NtA-Ag or biotinylated NtA-Ag-LAMα1-p1 co-stained with fluorophore-coupled streptavidin (red) and Hoechst DNA staining (blue). Scale bars = 10 μm.

Article Snippet: After washing, the pellet was incubated with MACS streptavidin beads solution (1:200, 130-048-101, Miltenyi Biotec) for 1 h on ice.

Techniques: In Vivo, Conjugation Assay, Western Blot, Injection, Immunostaining, Staining

Journal: iScience

Article Title: Identification of skeletal muscle stem cell adhesion motifs using spot-synthesis-based peptide arrays

doi: 10.1016/j.isci.2025.114498

Figure Lengend Snippet: Adhesion peptides as biosensors (A) Scheme shows the detection strategy of biotinylated candidate adhesion peptides to visualize their cell surface binding. (B) Fluorescent streptavidin detection (green) of the different biotinylated candidate peptides bound to myoblasts in 2D culture in combination with Hoechst DNA staining to visualize DNA (blue). Myoblasts not incubated with peptide (No peptide) or exposed to the scrambled peptide sequences (SCR-p5) are shown as a control. Scale bar = 5 μm. (C) Integrin β1 (Itg-β1) immunostaining (red) of muscle stem cells (MuSCs) on single muscle fibers after 48 h of culture in combination with the fluorophore-coupled streptavidin detection of the different biotinylated candidate peptides (green) and Hoechst DNA staining (blue). Single fibers not incubated with peptide (No peptide) as well as one of the scrambled peptide sequences (SCR-p5) are shown as a control. Scale bar = 2 μm.

Article Snippet: After washing, the pellet was incubated with MACS streptavidin beads solution (1:200, 130-048-101, Miltenyi Biotec) for 1 h on ice.

Techniques: Binding Assay, Staining, Incubation, Control, Immunostaining