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Applikationshinweise
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Western blotting, 1:1,000 for chemiluminescence detection system Immunoprecipitation, Not tested Immunohistochemistry, 1:1,000 Heat treatment is necessary for staining paraffin embedded sections. Autoclave, 125 o C for 5 minutes in 10 mM citrate buffer containing 0.05% Tween-20 (pH 6.0). Immunocytochemistry, 1:200 Flow cytometry, 1:200 (final concentration) . Protocol: Immunohistochemical staining for paraffin-embedded sections 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 Autoclave: Heat the slides immersed in retrieval solution [10 mM citrate buffer containing 0.05% Tween-20 (pH 6.0)] at 125oC for 5 minutes in pressure boiler. After boiling, the slides should remain in the pressure boiler until the temperature is cooled down to 80oC. Let the immersed slides further cool down at room temperature for 40 minutes. 5) 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. 6) Remove the slides from PBS, wipe gently around each section and cover tissues with 5% FCS in PBS for 30 minutes at room temperature to block non-specific 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 5% FCS as suggested in the APPLICATIONS. Note: It is essential for every laboratory to determine the optional titers of the primary antibody to obtain the best result. 8) Incubate the sections for 2 hours 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 ENVISION/HRP polymer reagent (DAKO, code no. K1491). Incubate for 15 minutes at room temperature. Wash as in step 9). 11) Visualize by reacting for 5 minutes with DAB substrate solution (DAKO, code no. K3465). *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 controls for Immunohistochemistry, kidney, pancreas) SDS-PAGE & Western Blotting 1) Wash the 2 x 10 6 cells 3 times with PBS and suspend with 100 uL of cold Lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS) 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. 3) Mix the sample with equal volume of Laemmli's sample buffer. 4) Incubate the samples for 1 hour at 37 o C and centrifuge at 10,000 x g for 5 minutes. Transfer the supernatant into a new tube. Load 10 uL 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 5% skimmed milk (in PBS, pH 7.2) for 2 hours at room temperature, or overnight at 4 o C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 2% skimmed milk as suggested in the APPLICATIONS for 2 hours 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 3 times). 9) Incubate the membrane with the 1:2,000 HRP-conjugated anti-mouse IgG (MBL, code no. 458) diluted with 2% 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) Drain 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 and develop the film as usual. The condition for exposure and development may vary. Immunocytochemistry 1) Culture the cells at an appropriate condition on a glass slide. (for example, spread 1x10 4 cells for one slide, then incubate in a CO 2 incubator for one night.) 2) Wash the cells 3 times with PBS. Western blot analysis of SLC7A8 expression in Myc-tagged SLC7A8 transfected 293T (2, 3) and parental cell (1) using BMP041 (1, 2) or anti-Myc-tag antibody (3, MBL, code no. M047-3). 1 2 3 kDa 150100 755037 25 1 2 3 kDa 150100 755037 25.
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Alternativen Western Blot (WB), Immunhistochemie (IHC), Immunzytochemie (ICC), Durchflusszytometrie (FACS)
