Selenium Binding Protein 1 (SELENBP1) (full length) Mouse Monoclonal Antibody [Clone ID: 4D4]

This product was originally produced by MBL International.

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 o 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 o C 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 μ L 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/cm 2 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 o C.
7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.)
8) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 6 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 (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 control for Western blotting; ZR75-1)

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) Remove the slides from the citrate buffer and cover each section with 3% H 2 O 2 for 10 minutes at room temperature to block endogenous peroxidase activity. Wash 3 times in PBS for 5 minutes each.
5) Remove the slides from PB S, wipe gently around each section and cover tissues with Protein Blocking Agent for 5 minutes to block non-specific staining. Do not wash.
6) Tip off the blocking buffer, wipe gently around each section and cover tissues with primary antibody diluted with PBS containing 1% BSA as suggest in the APPLICATIONS .
7) Incubate the sections for 1 hour at room temperature.
8) Wash the slides 3 times in PBS for 5 minutes each.
9) Wipe gently around each section and cover tissues with Polyvalent Biotinylated Antibody. Incubate for 10 minutes at room temperature. Wash as in step 8).
10) Wipe gently around each section and cover tissues with Streptavidin-Peroxidase (Ultrat ech HRP Kit). Incubate for 10 minutes at room temperature. Wash as in step 8).
11) Visualize by reacting for 10-20 minutes with substrate solution containing 7.5 mg DAB, 40 μ L of 30% H 2 O 2 in 150 mL PBS.
* DAB is a suspect carcinogen and must be handled with care. Always wear gloves.
12) Wash the slides in water for 5 minutes.
13) 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.
14) Now ready for mounting.
(Positive control for Immunohistochemistry; human primary lung adenocarcinoma)