VALIDATION IMAGES
Sample: Heart (Mouse) Lysate at 40 ug
Primary: Anti- GLUT4 (bs-0384R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/10000 dilution
Predicted band size: 54 kD
Observed band size: 54 kD
Sample:
Heart(Rat) Cell Lysate at 40 ug
Primary: Anti-GLUT4 (bs-0384R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 54 kD
Observed band size: 54 kD
Sample:
Lane 1: NIH/3T3 (Mouse) Cell Lysate at 30 ug
Lane 2: Heart (Mouse) Lysate at 40 ug
Lane 3: Heart (Rat) Lysate at 40 ug
Lane 4: Muscle (Mouse) Lysate at 40 ug
Lane 5: Muscle (Rat) Lysate at 40 ug
Primary: Anti-GLUT4 (bs-0384R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 51 kD
Observed band size: 51 kD
Tissue/cell: rat kidney tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-GLUT4 Polyclonal Antibody, Unconjugated(bs-0384R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Tissue/cell: rat kidney tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-GLUT4 Polyclonal Antibody, Unconjugated(bs-0384R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Tissue/cell: NIH/3T3 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (GLUT4) polyclonal Antibody, Unconjugated (bs-0384R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control (blue line): K562 (blue).
Primary Antibody (green line): Rabbit Anti-GLUT4 antibody (bs-0384R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 70% ethanol (overnight at 4℃) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.