MICA Mouse Monoclonal Antibody [Clone ID: BAMO3]

Immunoprecipitation
1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40) containing appropriate protease inhibitors. Incubate it at 4°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°C and transfer the supernatant to another tube.
3) Add primary antibody as suggest in the APPLICATIONS into 300 µL of cell extract. Mix well and incubate with gentle agitation for 30-120 minutes at 4°C.
4) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds).
5) Resuspend the agarose in 20 µL of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes.
6) Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis.
7) 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.
8) 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.
9) Incubate the membrane with 1 µg/mL of anti-MICA/B (BAMO1) monoclonal antibody diluted with PBS, pH 7.2 containing 1% skimmed for 1 hour at room temperature. (The concentration of antibody will depend on condition.)
10) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 3 times).
11) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
12) Wash the membrane with PBS-T (5 minutes x 3 times).
13) 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.
14) 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 control for Immunoprecipitation; HeLa)

Flow Cytometric analysis for floating cells
We usually use Fisher tubes or equivalents as reaction tubes for all step described below.
1) Wash the cells 3 times with washing buffer [PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3].
2) Resuspend the cells with washing buffer (5x10e6 cells/mL).
3) Add 50 µL of the cell suspension into each tube, and centrifuge at 500 x g for 1 minute at room temperature (20~25°C). Remove supernatant by careful aspiration.
4) Add 20 µL of Clear Back (human Fc receptor blocking reagent) to the cell pellet after tapping. Mix well and incubate for 5 minutes at room temperature.
5) Add 40 µL of the primary antibody at the concentration of as suggest in the APPLICATIONS diluted in the washing buffer. Mix well and incubate for 30 minutes at room temperature.
6) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature. Remove supernatant by careful aspiration.
7) Add 30 µL of 1:100 FITC conjugated anti-mouse IgG diluted with the washing buffer. Mix well and incubate for 15 minutes at room temperature.
8) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature. Remove supernatant by careful aspiration.
9) Resuspend the cells with 500 µL of the washing buffer and analyze by a flow cytometer.
(Positive controls for Flow cytometry; 293T, Jurkat, HeLa)

ELISA 
1) Distribute 100 µL/well of the anti-MICA (AMO1) or anti-MICB (BMO1) monoclonal antibody (1 µg/mL) diluted with PBS to each well.2) Incubate it overnight at 4°C.
3) Add 100 µL/well of 15% BSA/PBS.
4) Incubate it for 1 hour at 37oC.
5) Wash the plates 4 times with PBS-T [0.05% Tween-20 in PBS].
6) Distribute 100 µL/well of the samples or the recombinant MICA or MICB standard (0~20 ng/mL) diluted with 7.5% BSA/PBS to each well.
7) Incubate it for 2 hours at 37°C.
8) Wash the plates 4 times with PBS-T.
9) Distribute 100 µL/well of the anti-MICA/B monoclonal antibody (BAMO3) (1 µg/mL) to each well.
10) Incubate it for 2 hours at 37°C. 
11) Wash the plates 4 times with PBS-T.
12) Distribute 100 µL/well of the 1:5,000 or 1:2,000 HRP-conjugated anti-mouse IgG2a diluted with 3.75% BSA/PBS to each well.
13) Incubate it for 1 hour at 37°C.
14) Wash the plates 6 times with PBS-T.
15) Distribute 100 µL/well of the tetra-methylbenzidine (TMB) containing solution.
16) Incubate it for 5~60 minutes. The condition for reaction may vary.
17) Distribute 100 µL/well of 1 M H2SO4 to each well and stop enzyme reaction.
18) After gentle mixing, determine the absorbance at 450 nm of each well by a spectrophotometer.