Rb Antibody Sampler Kit
cat.: HAK21105
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Uniprot #: SwissProt: P06400 Human | P13405 Mouse | P33568 Rat | P06400 Human | P13405 Mouse | P06400 Human | P13405 Mouse | P33568 Rat | P06400 Human
Images
HAK21105_1.jpg Fig1: Western blot analysis of Phospho-Rb (S795) on different lysates with Rabbit anti-Phospho-Rb (S795) antibody (HA722764) at 1/2,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HeLa treated with 100ng/mL Nocodazole for 18 hours cell lysate (20 µg/Lane)
Lane 3: HeLa treated with 100ng/mL Nocodazole for 18 hours cell lysate, then the membrane treated with λpp for 1 hour (20 µg/Lane)

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

Exposure time: 3 minutes; 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 (HA722764) at 1/2,000 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.
HAK21105_2.jpg Fig2: Western blot analysis of Phospho-Rb (S807) on different lysates with Rabbit anti-Phospho-Rb (S807) antibody (ET1602-36) at 1/2,000 dilution.

Lane 1: K-562 cell lysate (no heat) (20 µg/Lane)
Lane 2: K-562 cell lysate (20 µg/Lane)
Lane 3: K-562 cell lysate (no heat), treated with λpp for 1 hour (20 µg/Lane)
Lane 4: K-562 cell lysate, treated with λpp for 1 hour (20 µg/Lane)

Notice: no heat means the lysate is not boiled.

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

Exposure time: 2 minutes;
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 (ET1602-36) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
HAK21105_3.jpg Fig3: Western blot analysis of Phospho-Rb (S780) on different lysates with Rabbit anti-Phospho-Rb (S780) antibody (HA721513) at 1/1,000 dilution.

Lane 1: Jurkat serum starved for 44 hours whole cell lysate
Lane 2: Jurkat serum starved for 24 hours then 10% FBS incubated for 20 hours whole cell lysate
Lane 3: Jurkat serum starved for 44 hours then treated with λpp for 1 hour whole cell lysate
Lane 4: Jurkat serum starved for 24 hours then 10% FBS incubated for 20 hours then treated with λpp for 1 hour whole cell lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 1 minute 59 seconds;

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 (HA721513) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
HAK21105_4.jpg Fig4: Western blot analysis of Phospho-Rb (S795) on different lysates with Rabbit anti-Phospho-Rb (S795) antibody (HA722764) at 1/2,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 100ng/mL Nocodazole for 18 hours cell lysate
Lane 3: U-2 OS cell lysate
Lane 4: U-2 OS treated with 50ng/mL Nocodazole for 8 hours cell lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time: Lane 1-4 (left): 40 seconds; Lane 1-4 (right): 1 minute; ECL: K1802;
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 (HA722764) at 1/2,000 dilution and competitor's antibody at 1/2,000 dilution were 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.
HAK21105_5.jpg Fig5: Immunocytochemistry analysis of HeLa cells treated with 100ng/mL Nocodazole for 18 hours labeling Phospho-Rb (S795) with Rabbit anti-Phospho-Rb (S795) antibody (HA722764) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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 (S795) antibody (HA722764) at 1/500 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 (M1305-2, 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.