[IF=0.85] Yabe et al. Characterization of SET/I2PP2A isoforms in dogs. (2014) J.Vet.Med.Sc. 76:1235-40  IF ;  Canine.  

PubMed:24897959

Sample:
Lane 1: Human HepG2 cell lysates
Lane 2: Human K562 cell lysates
Lane 3: Human MOLT4 cell lysates
Lane 4: Human HeLa cell lysates
Lane 5: Human A431 cell lysates
Primary: Anti-SET (bs-5943R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 32 kDa
Observed band size: 39,41 kDa

Hela 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 (SET) polyclonal Antibody, Unconjugated (bs-5943R) 1:25, 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):Molt4 (Black).
Primary Antibody (green line): Rabbit Anti-SET antibody (bs-5943R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647
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 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.