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Applikationshinweise
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Western blotting, 1:500 for chemiluminescence detection system Immunoprecipitation, Not tested Immunohistochemistry, 1:500 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, 1:100 Flow cytometry, 1:100 (final concentration) . Protocol: Immunoh www.mblintl.com sections 1) Deparaffinize the sections with Xylene 3 times for 3-5 e slides with Ethanol 3 times for 3-5 min 3 times for 3-5 minutes each. 4) H r cool down at room temperature for 4 dase activity. Wash 3 times i spect wed by es for 3 minutes each. 14) for Immunohistochemistry, human stomach, kidney) minutes each. 2) Wash th utes each. 3) Wash the slides with PBS Heat treatment eat 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 furthe 0 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 peroxi n 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 suggest in tAPPLICATIONS. he 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 su 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, folloimmersing in Xylene 3 timNow ready for mounting. (Positive control 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, inutes at ample with equal volume of Laemmli's samp rylamide gel for electrophoresis. the sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 m n 4 o C and transfer the supernatant to another tube. 3) Mix the s le 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-polyac 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 suggest 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. Western blot analysis of SLC9A1 expression in Myc-tagged SLC9A1 transfected 293T (1, 3) and parental cell (2) using BMP023 (2, 3) or anti-Myc-tag 120 kDa 97 55.4 1 2 3 SLC9A1 36.1 28.9 - - - - - 120 kDa 97 55.4 1 2 3 SLC9A1 36.1 28.9 - - - - - - - - - - Immunocytochemistry 1) Culture the cells in the appropriate condition on a glass slide. (for example, spread 1x10 4 cells for one slide, then e night.) incubate in a CO 2 incubator for on 2) Wash the cells 3 times with PBS. 3) Fix the cells by immersing the slide in PBS containing 4% antibody (1, MBL, code no. M047-3).
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