VALIDATION IMAGES
Sample: 293T (human)Cell Lysate at 40 ug
Primary: Anti- KLF4 (bs-1064R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 56 kD
Observed band size: 60 kD
Sample:
Lane 1: Mouse NIH/3T3 cell lysates
Lane 2: Human A431 cell lysates
Lane 3: Human LOVO cell lysates
Lane 4: Human HeLa cell lysates
Lane 5: Human MCF-7 cell lysates
Lane 6: Human A549 cell lysates
Primary: Anti-KLF4 (bs-1064R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 56 kDa
Observed band size: 50 kDa
Sample:
Hela Cell(Human)Lysate at 30 ug
Primary: Anti- KLF4 (bs-1064R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 56kD
Observed band size: 56kD
Paraformaldehyde-fixed, paraffin embedded (Rat lung); 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 (KLF4) Polyclonal Antibody, Unconjugated (bs-1064R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Tissue/cell: human colon carcinoma; 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-KLF4 Polyclonal Antibody, Unconjugated(bs-1064R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Blank control: MCF7(blue). Primary Antibody: Rabbit Anti-KLF4 AF647 Conjugated antibody (bs-1064R-AF647), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG/AF647(orange) ,used under the same conditions.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with ice-cold 90% methanol for 30 min on ice. The cells were washed twice with 1 X PBS. The cells were incubated in 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions followed by the incubated with antibody (bs-1064R-AF647, 1μg /1x10^6 cells) for 30 min on ice. Acquisition of 20,000 events was performed.
Blank control: K562.
Primary Antibody (green line): Rabbit Anti-KLF4 antibody (bs-1064R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 1μg /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.
