Rb (RB1) (612-928) Mouse Monoclonal Antibody [Clone ID: 3H9]

CAT#: AM26677AF-N

Rb (RB1) (612-928) mouse monoclonal antibody, clone 3H9, Azide Free


Product Images

Specifications

Product Data
Clone Name 3H9
Applications IHC, IP, WB
Recommended Dilution Western blot: 5-10 μg/ml.
Immunohistochemistry on paraffin sections: 5 μg/ml; Heat treatment is necessary. Microwave oven; 2 times for 10 minutes each in citrate buffer (pH 6.5).
Immunoprecipitation:  1-5 μg / 200-300 μl of cell extract.
For details see protocols below.
Reactivities Human
Host Mouse
Isotype IgG2a
Clonality Monoclonal
Immunogen Recombinant human Rb protein corresponding to amino acids 612-928
Specificity

This antibody reacts with retinoblastoma gene product (110-115 kDa).

Formulation PBS containing 50% glycerol, pH 7.2
State: Azide Free
State: Liquid Ig fraction without preservatives
Concentration lot specific
Purification Protein A Sepharose
Conjugation Unconjugated
Storage

Store at 2 - 8 °C.

Stability Shelf life: one year from despatch.

Background Mutation of the retinoblastoma tumor suppressor gene alone is sufficient to cause retinoblastoma in humans, suggesting that it might play a role in the normal coordination of cell proliferation and cell death. Deletion or mutational inactivation of the retinoblastoma tumor suppressor protein (Rb) is correlated with the genesis of a variety of human cancers including retinoblastoma, osteosarcoma, and carcinomas of the breast, bladder, and lung. Rb protein is phosphorylated by cyclinD-Cdk4/Cdk6 and cyclinA/cyclinE-Cdk2 during the G1/S transition. This phosphorylation causes the inactivation of the growth inhibitory functions of Rb. Rb undergoes phosphorylation and functional inactivation, permitting the cell to proceed into late G1.
Synonyms Retinoblastoma 1, Rb, p105-Rb, pRb, pp110, OSRC
Note This product was originally produced by MBL International.

Protocol:

SDS-PAGE & Western Blotting
1) Wash the cells 3 times with PBS and suspend with 10 volumes 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 oC 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 oC and transfer the supernatant to another tube. Measure the protein concentration of the supernatant and add the Lysis buffer to make an 8 mg/mL solution.
3) Mix the sample with an equal volume of Laemmli’s sample buffer.
4) Boil the samples for 2 minutes and centrifuge. Load 10 µL 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/cm2 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 specific transfer procedure.
6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4°C.
7) Incubate the membrane with primary antibody diluted with PBS, pH7.2 containing 1% skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The optimal concentration of antibody will depend on the experimental conditions.)
8) Wash the membrane with PBS (5 minutes x 6 times).
9) Incubate the membrane with the 1:10,000 POD-conjugated anti-mouse IgG diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
10) Wash the membrane with PBS (5 minutes x 6 times).
11) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap.
12) Expose to X-ray film in a dark room for 5 minutes. Develop the film as usual. The conditions for exposure and development may vary.

Immunoprecipitation
1) Wash the cells 3 times with PBS and suspend with 10 volumes 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 oC 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 oC and transfer the supernatant to another tube.
3) Add primary antibody as suggested in the APPLICATIONS into 250 µL of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4 oC. Add 20 µL of 50% Protein A-agarose beads resuspended in the Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4 oC.
4) Wash the beads 3-5 times with ice-cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds).
5) Resuspend the beads in 20 µL of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10 µL/lane for the SDS-PAGE analysis. (See SDS-PAGE & Western blotting.)

Immunohistochemical staining for paraffin-embedded sections: SAB method
1) Deparaffinize the sections with Xylene 3 times for 3-5 minutes each.
2) Wash the slides with Ethanol 3 times for 3-5 minutes each.
3) Wash the slides with PBS 3 times for 3-5 minutes each.
4) Heat treatment Heat treatment by microwave oven: Place the slides put on staining basket in 500 mL beaker with 500 mL citrate buffer (pH 6.5). Cover the beaker with plastic wrap, then process the slides 2 times for 10 minutes each at 500 W with microwave oven. Let the slides cool down in the beaker at room temperature for about 40 minutes.
5) Remove the slides from the citrate buffer and cover each section with 3% H2O2 for 10 minutes at room temperature to block endogenous peroxidase activity. Wash 3 times in PBS for 5 minutes each.
6) Remove the slides from PBS, wipe gently around each section and cover tissues with Protein Blocking Agent for 5 minutes to block non-specific antibody staining. Do not wash.
7) Tip off the blocking buffer, wipe gently around each section and cover tissues with primary antibody diluted with PBS containing 1% BSA as suggested in the APPLICATIONS.
8) Incubate the sections for 1 hour at room temperature.
9) Wash the slides 3 times in PBS for 5 minutes each.
10) Wipe gently around each section and cover tissues with Polyvalent Biotinylated Antibody (Ultratech HRP Kit). Incubate for 10 minutes at room temperature. Wash as in step 9.
11) Wipe gently around each section and cover tissues with Streptavidin-Peroxidase. Incubate for 10 minutes at room temperature. Wash as in step 9.
12) Visualize by reacting for 10-20 minutes with substrate solution containing 7.5 mg DAB, 40 µL of 30% H2O2 in 150 mL PBS. *DAB is a suspected carcinogen and must be handled with care. Always wear gloves.
13) Wash the slides in water for 5 minutes.
14) Counter stain in hematoxylin for 1 minute, wash the slides 3 times in water for 5 minutes each, and then immerse the slides in PBS for 5 minutes. Dehydrate by immersing in Ethanol 3 times for 3 minutes each, followed by immersing in Xylene 3 times for 3 minutes each.
15) Now ready for mounting.

Reference Data

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