Retinoblastoma 1 antibody (pSer780)

Catalog No. ABIN675294

Product Details anti-Retinoblastoma 1 Antibody

Target: Retinoblastoma 1 (RB1)

Binding Specificity: pSer780

Reactivity: Human, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This Retinoblastoma 1 antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Flow Cytometry (FACS), Immunohistochemistry (Frozen Sections) (IHC (fro))

Cross-Reactivity: Human, Rat

Predicted Reactivity: Mouse,Dog,Cow,Chicken

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic phosphopeptide derived from human Rb around the phosphorylation site of (Ser780)

Isotype: IgG

Application Details

Application Notes: WB 1:300-5000
ELISA 1:500-1000
FCM 1:20-100
IHC-P 1:200-400
IHC-F 1:100-500
IF(IHC-P) 1:50-200
IF(IHC-F) 1:50-200
IF(ICC) 1:50-200

Restrictions: For Research Use only

Independent Validation (WB)

Validation #029795 (Western Blotting)

by: Alamo Laboratories Inc

No.: #029795

Date: 08/21/2014

Antigen: Retinoblastoma 1 (RB1) (pSer780)

Lot Number: 120508

Method validated: Western Blotting

Positive Control: NIH/3T3 cells irradiated with 25 Joules/m2 UV-C

Negative Control: Non-irradiated NIH/3T3 cells

Notes: A strong band was observed in the positive control sample at the correct molecular weight. An additional band was also observed in the positive sample at a lower molecular weight which may represent non-specific binding. No bands were observed in the negative control sample.

Validation Images

Figure 1: Western Blot for RB1 pSer780. Grey arrowhead indicates the expected molecular weight of ~105 kDa.

Full Methods

Primary Antibody:

• Antigen: Retinoblastoma 1 (RB1) (pSer780)
• Catalog number: ABIN675294
• Lot number: 120508
• Antibody Dilution: 1:100

Secondary Antibody:

• Antigen: Goat Anti-Rabbit IgG (H + L)-HRP Conjugate
• Lot number: L170-6515
• Antibody Dilution: 1:10,000

Full Protocol:

• 1. The cell extracts were heated at 95°C for 5 minutes in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol.
• 2. 20 μL of heated culture-media were loaded and resolved on 8-16% SDS-polyacrylamide gel.
• 3. The Thermo Scientific - Spectra Multicolor Broad Range (Cat # 26634) were used as molecular mass markers.
• 4. Proteins were then transferred onto PVDF membrane by wet transfer and protein transfer was confirmed with Ponceau-S staining.
• 5. The PVDF membrane was incubated with 25 mL of blocking buffer [Tris Buffered Saline, pH 7.4 plus 0.1% TW20 (TBST)] containing 5% (W/V) BSA at room temperature for 1 hour.
• 6. The membrane was rinsed with TBST once.
• 7. The membrane was immersed with the protein side up in the primary antibody solution in TBST containing 5% (W/V) BSA and incubated for 20 hours at 4°C.
• 8. The membrane was rinsed in TBST thrice for 5 minutes each.
• 9. The membrane was incubated in the HRP-conjugated secondary antibody solution in TBST containing 5% (W/V) BSA and incubated for 1 hour at room temperature (~26°C) with gentle agitation.
• 10. The membrane was rinsed thrice TBST thrice for 5 minutes each.
• 11. The membrane was rinsed in TBS twice for 30 seconds each.
• 12. Signals were detected with ECL-2 Substrate. The blot was scanned for 50 minutes.
• 13. The membrane was rinsed three times TBST.
• 14. Incubated in Acidic Glycine Stripping Buffer at room temperature with gentle agitation for 3 times, 10 minutes each.
• 15. The membrane was washed in TBST 2 times for 10 minutes each.
• 16. Repeated Steps 5-12 with the loading control antibody (for Anti-actin) and its matching secondary antibody.

Experimental Notes: - No experimental challenges noted.

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: 0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.

Expiry Date: 12 months

References for anti-Retinoblastoma 1 antibody (ABIN675294)

Béguelin, Rivas, Calvo Fernández, Teater, Purwada, Redmond, Shen, Challman, Elemento, Singh, Melnick: "EZH2 enables germinal centre formation through epigenetic silencing of CDKN1A and an Rb-E2F1 feedback loop." in: Nature communications, Vol. 8, Issue 1, pp. 877, (2018) (PubMed).

Target Details for Retinoblastoma 1

Target: Retinoblastoma 1 (RB1)

Alternative Name: Rb/p105-Rb (RB1 Products)

Synonyms: OSRC antibody, RB antibody, p105-Rb antibody, pRb antibody, pp110 antibody, Rb antibody, Rb-1 antibody, P105-RB antibody, PP110 antibody, RB11 antibody, zgc:154147 antibody, RB transcriptional corepressor 1 antibody, RB transcriptional corepressor like 1 antibody, retinoblastoma 1 antibody, RB1 antibody, RBL1 antibody, Rb1 antibody, rb1 antibody

Background:

Synonyms: RbSer780, OSRC, P105 RB, P105RB, PP105, PP110, pRb, RB 1, RB1, RB1 protein, Retinoblastoma 1 including osteosarcoma, Retinoblastoma 1, Retinoblastoma associated protein, Including osteosarcoma, Osteosarcoma, p105-Rb, Rb, RB_HUMAN, Retinoblastoma suspectibility protein, Retinoblastoma-associated protein, Retinoblastoma related osteosarcoma, Retinoblastoma susceptibility gene.

Background: Nuclear Marker.The protein encoded by this gene is a negative regulator of the cell cycle and was the first tumor suppressor gene found. The encoded protein also stabilizes constitutive heterochromatin to maintain the overall chromatin structure. The active, hypophosphorylated form of the protein binds transcription factor E2F1. Defects in this gene are a cause of childhood cancer retinoblastoma (RB), bladder cancer, and osteogenic sarcoma.

Gene ID: 5925

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