Sample:
MCF-7(Human) Cell Lysate at 30 ug
Primary: Anti-FAK (SL1340R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 116 kD
Observed band size: 116 kD
Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 ug
Lane 2: NIH/3T3 (Mouse) Cell Lysate at 30 ug
Lane 3: Cerebrum (Rat) Lysate at 40 ug
Lane 4: 293T (Human) Cell Lysate at 30 ug
Lane 5: A431 (Human) Cell Lysate at 30 ug
Lane 6: Huvec (Human) Cell Lysate at 30 ug
Lane 7: A549 (Human) Cell Lysate at 30 ug
Primary: Anti-FAK (SL1340R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 125 kD
Observed band size: 120 kD
Sample:Thymus (Mouse) Lysate at 40 ug
Primary: Anti-FAK(SL1340R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 116kD
Observed band size: 116kD
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 (FAK) Polyclonal Antibody, Unconjugated (SL1340R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
HUVEC cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (FAK) polyclonal Antibody, Unconjugated (SL1340R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Tissue/cell: mouse lung tissue;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-FAK/PTK2 Polyclonal Antibody, Unconjugated(SL1340R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(SL0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,SLC0033) was used to stain the cell nuclei
Blank control (blue line): Hep G2 (blue).
Primary Antibody (green line): Rabbit Anti-FAK antibody (SL1340R)
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 (Overmight at 4℃) and then permeabilized with 90% ice-cold methanol for 30 min at -20℃.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.
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