[IF=6.59] Choi, Sik‐Won, et al. "Repositioning Potential of PAK4 to Osteoclastic Bone Resorption." Journal of Bone and Mineral Research (2015).  WB ;  Mouse.  

PubMed:25640698

Sample:
Lane 1: Human PANC-1 cell lysates
Lane 2: Human A431 cell lysates
Lane 3: Human A549 cell lysates
Lane 4: Human MCF-7 cell lysates
Primary: Anti-Phospho-PAK6 (Ser560) (bs-3314R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 75 kDa
Observed band size: 73 kDa

Sample:
Spleen (Mouse) Lysate at 40 ug
Primary: Anti-Phospho-PAK6(Ser560) (bs-3314R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 75 kD
Observed band size: 73 kD

Paraformaldehyde-fixed, paraffin embedded (mouse esophagus); 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 (Phospho-PAK6 (Ser560)) Polyclonal Antibody, Unconjugated (bs-3314R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse skin); 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 (Phospho-PAK6 (Ser560)) Polyclonal Antibody, Unconjugated (bs-3314R) at 1:100 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 (Phospho-PAK6 (Ser560)) Polyclonal Antibody, Unconjugated (bs-3314R) at 1:100 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 (Phospho-PAK6 (Ser560)) Polyclonal Antibody, Unconjugated (bs-3314R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat skin); 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 (Phospho-PAK6 (Ser560)) Polyclonal Antibody, Unconjugated (bs-3314R) at 1:100 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

A431 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 (Phospho-PAK6 (Ser560)) polyclonal Antibody, Unconjugated (bs-3314R) 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.

Blank control（black line）:A549.
Primary Antibody (green line): Rabbit Anti-Phospho-PAK6 (Ser560) antibody (bs-3314R)
Dilution:1ug/Test;
Secondary Antibody（white blue line）: Goat anti-rabbit IgG-AF488
Dilution: 0.5ug/Test.
Isotype control（orange line）: Normal Rabbit IgG
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.