VALIDATION IMAGES
Sample:
Brain (mouse) Lysate at 40 ug
Heart (mouse) Lysate at 40 ug
Primary: Anti- beta tubulin(bs-4511R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 55 kD
Observed band size: 55 kD
Sample:
Brain (Rat) Lysate at 40 ug
Heart (Rat) Lysate at 40 ug
Primary: Anti-Beta tubulin (bs-4511R) at 1/300 dilution
Secondary: HRP conjugated Goat-Anti-rabbit IgG (bs-0295G-HRP) at 1/5000 dilution
Predicted band size: 55 kD
Observed band size: 50 kD
Sample:
Cerebrum (Rat) Lysate at 40 ug
Cerebrum (Mouse) Lysate at 40 ug
Primary: Anti- Beta tubulin (bs-4511R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 55 kD
Observed band size: 55 kD
Sample:
Cerebrum (Mouse)Cell Lysate at 40 ug
Primary: Anti-Beta tubulin (bs-4511R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 55 kD
Observed band size: 55kD
Sample:
Lane 1: Mouse Cerebrum tissue lysates
Lane 2: Mouse Cerebellum tissue lysates
Lane 3: Human SH-SY5Y cell lysates
Lane 4: Human HeLa cell lysates
Primary: Anti-Beta tubulin (bs-4511R) at 1/2000 dilution
Secondary: HRP conjugated Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 50 kDa
Observed band size: 48 kDa
Sample:
Lane 1,3,5: Mouse Cerebellum tissue lysates
Lane 2,4,6: Rat Cerebellum tissue lysates
Primary: Anti-Beta tubulin (bs-4511R) at 1/10000~1/50000 dilution
Secondary: HRP conjugated Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 50 kDa
Observed band size: 48 kDa
Sample:
Lane 1: Mouse Cerebrum tissue lysates
Lane 2: Rat Cerebrum tissue lysates
Primary: Anti-Beta tubulin/HRP (bs-4511R-HRP) at 1/20000 dilution
Predicted band size: 55 kD
Observed band size: 52 kD
Sample:
Heart (Mouse) Lysate at 40 ug
MCF-7 Cell (Human) Lysate at 40 ug
Primary: Anti-Beta tubulin (bs-4511R) at 1/300 dilution
Secondary: HRP conjugated Goat-Anti-rabbit IgG (bs-0295G-HRP) at 1/5000 dilution
Predicted band size: 55 kD
Observed band size: 55 kD
Sample: Spleen (Mouse) Lysate at 40 ug
Primary: Anti- Beta tubulin (bs-4511R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 55 kD
Observed band size: 55 kD
Paraformaldehyde-fixed, paraffin embedded (mouse kidney); 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; Incubation with (Beta tubulin (Loading Control)) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human kidney); 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; Incubation with (Beta tubulin (Loading Control)) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 tubulin (Loading Control)) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat cerebellum); 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 tubulin (Loading Control)) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Mouse brain); 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 tubulin ) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 tubulin(Loading Control)) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse brain); 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 tubulin(Loading Control)) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat brain); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat ovary); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat brain); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat ovary); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse brain); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse ovary); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat heart); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse ovary); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat pancreas); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse brain); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse heart); 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 tubulin) Polyclonal Antibody, Unconjugated (bs-4511R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Blank control:HL-60.
Primary Antibody (green line): Rabbit Anti-Beta tubulin antibody (bs-4511R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF488
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 0.1%PBST for 20 min at room temperature. 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.