Western (Immuno-) Blotting Protocol[Santa Cruz] -Western-生物通

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Sample prepare

:: MONOLAYER CELLS

Remove culture medium and rinse a subconfluent, 100 mm cell culture plate with PBS at room temperature. The following steps should be performed on ice or at 4?C using fresh, ice cold buffers.
Add 0.6 ml of RIPA buffer (sc-24948) to a 100 mm cell culture plate. Gently rock plates for 15 minutes at 4?C. Scrape adherent cells with a cell scraper. Transfer the scraped lysate to a microcentrifuge tube.
Wash the plate once with 0.3 ml of RIPA buffer and combine with first lysate. (Optional: Add 10 祃 of 10 mg/ml PMSF (cat # sc-3597) stock and/or pass through a 21-gauge needle to shear the DNA.) Incubate 30?0 minutes on ice.
Microcentrifuge cell lysate at 10,000xg for 10 minutes at 4?C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet.

NOTE: For phosphorylation studies, lysates can be enriched for phosphoproteins using Santa Cruz Biotechnology Inc.磗 PhosphoCruz橮rotein Purification System (sc-24964).

:: SUSPENSION CELLS

Collect approximately 2.0 x 107 cells by low-speed centrifugation at room temperature for 5 minutes. Carefully remove culture medium.
Wash the pellet with PBS at room temperature, and again collect by low-speed centrifugation. Carefully remove supernatant.
Add 1.0 ml of ice cold RIPA buffer (sc-24948) with freshly added (Protease Inhibitors) and/or (Phosphatase Inhibitors). Gently resuspend cells in RIPA buffer with a pipet and incubate on ice for 30 minutes.
Further disrupt and homogenize cells by passing through 21-gauge needle, dounce homogenization or sonication, taking care not to raise the temperature of the lysate. (Optional: Add 10 祃 of 10 mg/ml PMSF stock; sc-3597) Incubate 30 minutes on ice.
Transfer to microcentrifuge tube(s) and centrifuge at 10,000xg for 10 minutes at 4?C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet

NOTE: For phosphorylation studies, lysates can be enriched for phosphoproteins using Santa Cruz Biotechnology Inc.磗 PhosphoCruz?Protein Purification System (sc-24964).

:: TISSUE SAMPLES

Weigh tissue and dice into very small pieces using a clean razor blade. Frozen tissue can be sliced very thinly and thawed in RIPA buffer (sc-24948) containing (Protease Inhibitors) and/or (Phosphatase Inhibitors). Use 3 ml of ice cold RIPA buffer per gram of tissue.
Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator, maintaining temperature at 4?C throughout all procedures. (Optional: Add 30 祃 of 10 mg/ml PMSF (sc-3597) stock per gram of tissue.) Incubate on ice for 30 minutes.
Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 minutes at 4?C. Remove supernatant and centrifuge again. The super-natant fluid is the total cell lysate. A longer centrifugation may be necessary to obtain a clear lysate.NOTE:
For phosphorylation studies, lysates can be enriched for phosphoproteins using Santa Cruz Biotechnology, Inc.磗 PhosphoCruz?Protein Purification System (sc-24964).

Elecroploresis

Mix sample (40?0 礸 whole cell lysate, 10?0 礸 nuclear extract or 10?0 ng purified protein per lane) with an equal volume of 2x electrophoresis sample buffer (sc-24945) and boil for 2? minutes. Unused samples may be stored at -20?C.?BR>Load up to 10 祃 of lysate per 1.0 mm of well width for gels of 0.75 mm thickness.
We recommend the use of Cruz Marker?molecular weight standards (sc-2035). Load 2 祃/well for 0.75 mm gels and 5 祃/well for 1.5 mm gels. When used with Cruz Marker?compatible secondary antibodies, internal standard bands will appear when the probed blot is exposed to detection reagent. Alternatively, use Prestained Molecular Weight Standards (sc-2361).
Electrophorese according to standard protocols.
Transfer proteins from the gel to a nitrocellulose or PVDF membrane (Western Blotting Membranes) using an electroblotting apparatus according to the manufacturer磗 protocols.

NOTE: Ready-made blots of human or mouse whole cell extracts or nuclear extracts or mouse tissues are available as Cruz Blot Systems?for Western Blotting.

Transfer

Block non-specific binding by incubating membrane in Blotto (either Blotto A or Blotto B, Blocking Reagents for Western Blotting Applications; BSA is recommended when using anti-bovine secondary antibodies) for 30?0 minutes at room temperature. Alternatively, the membrane may be blocked at 4?C overnight in a covered container, using Blotto without Tween-20.
Alternatively, if using a phospho-specific antibody, add 50 mM NaF (NaF: sc-24988) to the blocking solution and the antibody diluent to inhibit phosphatases.
Incubate the blocked membrane in primary antibody diluted in Blotto for 1 hour at room temperature. (For phospho-specific antibodies: Use Blotto with 50 mM NaF as diluent.) Optimal antibody concentration should be determined by titration. We recommend a starting dilution of 0.5?.0 礸/ml. Wash membrane three times for 5 minutes each with TBST (Buffers and General Solutions).
Incubate the membrane for 45 minutes at room temperature with horseradish peroxidase (HRP) conjugated secondary antibody, or alkaline phosphatase (AP) conjugated secondary antibody (Conventional Secondary Antibodies for Western Blotting), diluted to 1:500?:2000 in Blotto. If high backgrounds are observed, secondary antibody should be diluted further (up to 1:20,000). If Cruz Marker?molecular weight standards (sc-2035) are used in the gel, the Cruz Marker?compatible secondary antibodies must be used in order to visualize standards with ECL.
Wash membrane three times for 5 minutes each with TBST and once for 5 minutes with TBS (Buffers and General Solutions).
Incubate membrane in Chemiluminescence Luminol Reagent (sc-2048) according to Luminol data sheet, or visualize proteins using standard protocols. If luminol is used for visualization, an HRP-conjugated secondary antibody must be used.