RNF2 Mouse Monoclonal Antibody [Clone ID: 3-3]

Western blot: 1 μg/m.
Immunoprecipitation: 1-5 μg / 200-300 μl of cell extract.
For details see protocols below.
Not recommended for Immunohistochemistry.

This antibody reacts with Ring1B.

Store undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer.
Avoid repeated freezing and thawing.

Polycomb-group (PcG) proteins form multimeric complexes that maintain the state of transcriptional repression of several regulatory genes during development. Ring1B/Rnf2 forms part of a protein complex containing other PcG proteins, such as Mel18, Bmi1, MBLR, MPc3, and the spliceosome protein Sap155, and these complexes associate with chromatin to regulate transcription. Ring1B may also play a role in the regulation of Hox gene expression by PcG complexes. Deletion of Ring1B activity results in gastrulation arrest and cell cycle inhibition.

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 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 8 mg/mL solution.
3) Mix the sample with 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 precise 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 the anti-Ring1B monoclonal antibody (1 µg/mL) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
8) Wash the membrane with PBS (5 minutes x 6 times).
9) Incubate the membrane with the 1:10000 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 on the membrane, then incubate it with appropriate chemiluminescence 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 5 minutes. Develop the film as usual. The condition for exposure and development may vary.
Positive controls for Western blotting: Jurkat, NIH/3T3, CHO, BHK

Immunoprecipitation
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 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 1-5 µg of the anti-Ring1B monoclonal antibody 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.)
Positive control for immunoprecipitation: U937