![]() NOTE: LumiGLO ® substrate can be further diluted if signal response is too fast. Incubate membrane with 10 ml LumiGLO ® (0.5 ml 20X LumiGLO®, 0.5 ml 20X Peroxide and 9.0 ml Milli-Q water) with gentle agitation for 1 minute at room temperature.Incubate membrane with appropriate HRP-conjugated secondary antibody (1:2000) and HRP-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hour at room temperature.Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4☌.Wash three times for 5 minutes each with 15 ml of TBS/T.Incubate membrane in 25 ml of blocking buffer for 1 hour at room temperature.(Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature.NOTE: Volumes are for 10 cm x 10 cm (100 cm 2) of membrane for different sized membranes, adjust volumes accordingly. Membrane Blocking and Antibody Incubations Electrotransfer to nitrocellulose or PVDF membrane.Ĭ.NOTE: CST recommends loading prestained molecular weight marker ( #7720, 10 μl/lane) to verify electrotransfer and biotinylated protein ladder ( #7727, 10 μl/lane) to determine molecular weights. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm).Heat a 20 μl sample to 95–100☌ for 5 minutes cool on ice.Sonicate for 10–15 seconds for complete cell lysis and to shear DNA (to reduce sample viscosity).Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Lyse cells by adding 1X SDS sample buffer (100 μl per well of 6-well plate or 500 μl per plate of 10 cm diameter plate).Aspirate media from cultures wash cells with 1X PBS aspirate.Treat cells by adding fresh media containing regulator for desired time.PVDF membranes may also be used.Ī general protocol for sample preparation is described below. ![]() ![]() Blotting Membrane: This protocol has been optimized for nitrocellulose membranes, which CST recommends.Biotinylated Protein Ladder Detection Pack: ( #7727).Prestained Protein Marker, Broad Range (Premixed Format): ( #7720).Phototope ® -HRP Western Blot Detection System: ( #7071 anti-rabbit) or ( #7072 anti-mouse) Includes biotinylated protein ladder, secondary ( #7074 anti-rabbit) or ( #7076 anti-mouse) antibody conjugated to horseradish peroxidase (HRP), anti-biotin antibody conjugated to HRP, LumiGLO ® chemiluminescent reagent and peroxide.Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 with 5% nonfat dry milk for 20 ml, add 2 ml 10X TBS to 18 ml water, mix.Blocking Buffer: 1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk for 150 ml, add 15 ml 10X TBS to 135 ml water, mix.Nonfat Dry Milk: ( #9999) (weight to volume ).10X Tris Buffered Saline (TBS): ( #9997) To prepare 1 liter of 10X TBS: 24.2 g Tris base, 80 g NaCl adjust pH to 7.6 with HCl (use at 1X).Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5).NOTE: Prepare solutions with Milli-Q or equivalently purified water. Products available from Cell Signaling Technology are linked by their respective catalog numbers. For Western blots, incubate membrane with diluted antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween-20 at 4☌ with gentle shaking, overnight.
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