Retinoblastoma 1 antibody (pSer780)

Catalog No. ABIN5542077

The Mouse Monoclonal anti-Retinoblastoma 1 antibody is suitable to detect Retinoblastoma 1 in samples from Human. It has been validated for WB and EIA.

Quick Overview for Retinoblastoma 1 antibody (pSer780) (ABIN5542077)

Target: Retinoblastoma 1 (RB1)

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This Retinoblastoma 1 antibody is un-conjugated

Application: Western Blotting (WB), Enzyme Immunoassay (EIA)

Clone: 2C4

Product Details anti-Retinoblastoma 1 Antibody

Binding Specificity: AA 774-786, pSer780

Cross-Reactivity (Details): Does not react with Human 5637 (ATCC HTB9, RB mRNA negative) or Saos2 (ATCC HTB85 RB mRNA negative), and not with Mouse WR1-Rb-1, NIH/3T3, Ba/F3.

Purification: Protein A agarose

Immunogen: Synthetic human phospho-RB (Ser780) peptide, TRPPTLS (PO3) PIPHIP, which corresponding to amino acids 774-786 of human pRB

Isotype: IgG1

Application Details

Application Notes: Western blot: 1 μg/mL for chemiluminescence detection system. For details see protocol below.

Protocol: SDS-PAGE & Western Blotting 1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the cold Lysis buffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli's sample buffer. 4) Boil the samples for 3 minutes and centrifuge. Load 10 μ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20 % MeOH). See the manufacture's manual for precise transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 5 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 5 % skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.) 8) Wash the membrane with PBS-T [0.05 % Tween-20 in PBS] (5 minutes x 3 times). 9) Incubate the membrane with the 1:5,000 HRP-conjugated anti-mouse IgG diluted with 5 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS-T (10 minutes x 3 times). 11) Wipe excess buffer on the membrane, then incubate it with appropriate chemilumine scence reagent for 1 minute. Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 12) Expose to an X-ray film in a dark room for 3 minutes. Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting Jurkat, U937, MOLT4)

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. No preservative is contained.

Preservative: Azide free

Storage: -20 °C

Storage Comment: Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Target Details for Retinoblastoma 1

Target: Retinoblastoma 1 (RB1)

Alternative Name: retinoblastoma-associated protein,rb1

Background: Cyclin-dependent kinases (Cdks) play important roles in the regulation of the cell cycle. RB protein (pRB) is phosphorylated by cyclin D-Cdk4/Cdk6 and cyclin A/cyclin E-Cdk2 during the G 1 /S transition. This phosphorylation causes the inactivation of the growth inhibitory functions of pRB. pRB undergo phosphorylation and attendant functional inactivation, the cell proceed into late G 1. Cyclin D-Cdk4 specifically phosphorylates Ser780 in pRB, while cyclin E-Cdk2 and cyclin A-Cdk2 does not.

UniProt: P06400

Pathways: Cell Division Cycle, Intracellular Steroid Hormone Receptor Signaling Pathway, Mitotic G1-G1/S Phases, DNA Replication, Maintenance of Protein Location, Synthesis of DNA, Autophagy