Phospho-Rb (S608) Recombinant Rabbit Monoclonal Antibody [PSH11-04]

☑ Cell treatment (CT)

Western blot analysis of Phospho-Rb (S608) on different lysates with Rabbit anti-Phospho-Rb (S608) antibody (HA723271) at 1/5,000 dilution.

Lane 1: K-562 cell lysate (no heat)
Lane 2: SH-SY5Y cell lysate (no heat)
Lane 3: MCF7 cell lysate (no heat)
Lane 4: COS-1 cell lysate (no heat)
Lane 5: K-562 cell lysate (no heat), the membrane treated with λpp for 1 hour

Notice: no heat means the lysate is not boiled.

Lysates/proteins at 20 µg/Lane.

Predicted band size: 106 kDa
Observed band size: 110 kDa

Exposure time: 12 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723271) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of K-562 cells labeling Phospho-Rb (S608) with Rabbit anti-Phospho-Rb (S608) antibody (HA723271) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Rb (S608) antibody (HA723271) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187 , red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.