AM26673AF-N MAP2K1 antibody

Swiss Prot Num:

Q02750

Immunogen:

Recombinant Xenopus MAPKK

Genename:

5604

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 4oC 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 4oC 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 µL of sample per lane on a 1-mm-thick SDS-polyacrylamide gel and carry out 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 manufacturer'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 4oC.
7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on the conditions.)
8) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 3 times).
9) Incubate the membrane with the 1:10,000 HRP-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-T (10 minutes x 3 times).
11) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute.
12) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
13) Expose to an X-ray film in a dark room for 3 minutes.
14) Develop the film as usual. The condition for exposure and development may vary.
(Positive controls for Western blotting; Jurkat, Raji, HeLa, NIH/3T3, PC12)

Immunoprecipitation
1) Collect the cultured cells from 75-cm2 flask (containing about 0.5-1 x 10e7 cells).
2) Wash the cells 2 times with PBS and suspend with 1,200 ?L of cold Lysis buffer (50 mM Tris-HCl pH 7.4, 250mM NaCl, 0.1% NP-40, 2mM EDTA) containing appropriate protease inhibitors. Incubate it at 4oC with rotating for 15 minutes, then sonicate briefly (up to 10 seconds).
3) Centrifuge the tube at 12,000 x g for 10 minutes at 4oC and transfer the supernatant to another tube.
4) Add 50 ?L of 50% protein A agarose beads in the supernatant. Incubate it at 4oC with rotating for 60 minutes.
5) Centrifuge the tube at 12,000 x g for 5 minutes at 4oC. Supernatant is equally divided into another two tube.
6) Add primary antibody as suggested in the APPLICATIONS into the supernatant. Vortex briefly and incubate with gently agitation for 60-120 minutes at 4oC.
7) Add 20 µL of 50% protein A agarose beads into the tube. Mix well and incubate with gentle agitation for 30-60 minutes at 4oC.
8) Wash the beads 3-5 times with ice-cold IP buffer (10 mM Tris-HCl, 250 mM NaCl, 0.1% NP-40, pH7.4) (centrifuge the tube at 2,500 x g for 10 seconds).
9) Resuspend the beads in 30 µL of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Load 15 µL of the sample per lane in a 1-mm-thick SDS-polyacrylamide gel and carry out electrophoresis.
10) 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 manufacturer's manual for precise transfer procedure.
11) 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 4oC.
12) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on the conditions.)
13) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 3 times).
14) Incubate the membrane with the 1:10,000 HRP-conjugated anti-mouse IgG kappa chain diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
15) Wash the membrane with PBS-T (10 minutes x 3 times).
16) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute.
17) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
18) Expose to an X-ray film in a dark room for 3 minutes.
19) Develop the film as usual. The condition for exposure and development may vary.
(Positive control for Immunoprecipitation; Jurkat)

1) Miura, K., et al., J. Immunol. 167, 7027-7037 (2001).
2) Miura, K., et al., Blood 96, 2199-2205 (2000).
3) Fukuda, M., et al., J. Biol. Chem. 271, 20024-20028 (1996).
4) Fukuda, M., et al., J. Biol. Chem. 269, 33097-33101 (1994).
5) Kosako, H., et al., EMBO J. 13, 2131-2138 (1994).
6) Otsu, M., et al., FEBS Lett. 320, 246-250 (1993).
7) Kosako, H., et al., EMBO J. 12, 787-794 (1993).

Store (in aliquots) at -20 °C. Avoid repeated freezing and thawing.
Shelf life: one year from despatch.

Purification:

Protein A agarose

Buffer System:

PBS containing 50% glycerol

State:

Liquid Ig fraction without preservatives
Azide Free

Species reactivity (tested):

Human, mouse, rat, xenopus

Specificity:

This antibody reacts with MAPKK (MEK1).