VALIDATION IMAGES
Sample:
293T Cell (Human) Lysate at 40 ug
NIH/3T3 Cell (Mouse) Lysate at 40 ug
Muscle (Mouse) Lysate at 40 ug
Primary: Anti-β-Actin (bsm-33036M) at 1/5000 dilution
Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Sample:
Hela(Human) Cell Lysate at 30 ug
NIH/3T3(Mouse) Cell Lysate at 30 ug
A431(Human) Cell Lysate at 30 ug
Primary: Anti-beta-Actin (bsm-33036M) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Sample:
NIH/3T3 (Mouse) Cell Lysate at 30 ug
Hela (Human) Cell Lysate at 30 ug
A549 (Human) Cell Lysate at 30 ug
Primary: Anti-beta-Actin (bsm-33036M) at 1/1000~1/10000 dilution
Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Sample:
Cerebrum(Mouse) Lysate at 40 ug
Lung(Rat) Lysate at 40 ug
Primary: Anti-beta-Actin (bsm-33036M) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Sample:
A549(Human) Cell Lysate at 30 ug
Primary: Anti-beta-Actin (bsm-33036M) at 1/500 dilution
Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Sample:
Lovo(Human) Cell Lysate at 30 ug
Primary: Anti- beta-Actin (bsm-33036M) at 1/500 dilution
Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 46 kD
Sample:
Lane 1: Hela (Human) Cell Lysate at 30 ug
Lane 2: NIH/3T3 (Mouse) Cell Lysate at 30 ug
Lane 3: Lung (Rat) Lysate at 40 ug
Lane 4: Lung (Mouse) Lysate at 40 ug
Lane 5: MCF-7 (Human) Cell Lysate at 30 ug
Lane 6: A431 (Human) Cell Lysate at 30 ug
Lane 7: Stomach (Rat) Lysate at 40 ug
Lane 8: Stomach (Mouse) Lysate at 40 ug
Lane 9: Cerebrum (Rat) Lysate at 40 ug
Lane 10: Cerebrum (Mouse) Lysate at 40 ug
Primary: Anti-beta-Actin (bsm-33036M) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Paraformaldehyde-fixed, paraffin embedded (rat colon); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (beta-Actin (Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33036M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse stomach tissue); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (beta-Actin) Monoclonal Antibody, Unconjugated (ascites of bsm-33036M) at 1:2000 overnight at 4°C, followed by a conjugated secondary (sp-0024) for 20 minutes and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse colon); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (beta-Actin (Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33036M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Blank control:Hela.
Primary Antibody (green line): Mouse Anti-beta-Actin antibody (bsm-33036M)
Dilution: 1ug/Test;
Secondary Antibody : Goat anti-Mouse IgG-FITC
Dilution: 0.5ug/Test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃.The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.