Wash cells in the dish once with ice-cold PBS and aspirate off PBS. Add ice-cold NP40 Cell Lysis Buffer (RIPA Lysis Buffer: 25mM Tris-HCl pH7.5, 150mM NaCl, 1% NP-40, 1mM EDTA pH8.0. Add fresh: 1 mM PMSF, 1 mM Na3VO4, and 1 X Protease Inhibitor Cocktail-P2714, Sigma).
We recommend adding 0.8 to 1 ml NP40 Cell Lysis buffer for 10-cm cell culture dish, when cells are over 70% confluent.
For adherent cells, scrap cells using cell scraper (Fisherbrand Disposable Cell Lifter, Fisher Scientific, 08-773-1) under ice. For suspension cells, pellet the cells, then resuspend in lysis buffer under ice.
Transfer the cell lysis solution into eppendorf tubes after pipetting the cell pellets 15 times.
Sonicate the cell lysates under ice for 10 sec.
Centrifuge the lysate at 12,000g in a pre-cooled centrifuge for 15 minutes.
Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet.
Determine the protein concentration by any commercially available reagent kit. We recommend using BCA Protein Assay kit (Thermo Scientific, 23227).
At this step, the sample can be divided into aliquots (for example, 100ug per tube) and stored at –80o C for long-term.
Sample Preparation, Electrophoresis and Protein Transferring:
Add 5 ?g of the above prepared cell lysate into SDS Sample Buffer in a volume of total 10 ?l (for a mini-gel, up to 15 ug of protein can be loaded per lane). Use the SDS Sample Buffer, which is included in the over-expression cell lysate package.
Boil the SDS samples for 10 min before loading (Note: For membrane proteins, SDS samples should be incubated at room temperature for 30 minutes before loading.). Prepare a pre-stained protein standard (SeeBlue Plus 2 Prestained Standard, Invitrogen, LC5925) as well. We suggest adding an OriGene MYC/DDK Tagged Western Blot Molecular Weight Markers (MWM1001) if anti-tag antibodies are used for Western blot experiments. 1ug of MWM1001 is good for one loading.
Run the samples on a pre-cast SDS polyacrylamide gel at 170V (constant voltage) for 40 to 60 minutes until the dye reaches the bottom the gel. We recommend using NuPage 4-12% Bis-Tris gel (Invitrogen, NP0323BOX) for proteins with molecular weights smaller than 200 kD.
Remove the gel and soak in 1L of protein transfer buffer (made by mixing Tris base 48.5 g, Glycine 240.2 g, Methanol 3.2 L, 10N NaOH 1.5 ml, add water to 16 L) for 15 minutes. Cut the nitrocellulose membrane (ISC BioExpress, F-3139-3) to the similar size of the transfer area of the gel. Assemble the electroblotting cassette and place the electrodes in the blotting unit, according to the manufacture's instructions.
Transfer in Tris-Glycine transfer buffer at 100 V for 1 hour at constant current (not to exceed 0.4 A).
Following transfer, remove the membrane from the blotting cassette and mark the orientation of the gel with a pencil. Rinse briefly with PBS.
Wash the membrane with TBST (10mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) once for 5 min at room temperature. Block non-specific binding on the membrane with freshly prepared 5% nonfat dried milk for 1 hour on a shaking platform at room temperature.
Incubate the membrane with a specific primary antibody diluted in TBST and 5% nonfat dried milk at the manufacturer's recommended dilution with gentle agitation at 4o C overnight.
Wash three times for 5 min each with TBST.
Incubate with OriGene's HRP-conjugated secondary antibody (TA100015) at 1:5,000 in TBST-5% nonfat dried milk for 1 hour at room temperature.
Wash three times again for 5 minutes each with TBST.
For detection, use OriGene's Western Blot Luminol Reagent (TA100016) and prepare according to instructions.
Lay the membrane on a plastic surface with the protein side up. Add the mixed detection solution to the membrane. Incubate for 1 minute. Remove the excess solution and cover the membrane with transparent plastic.
Place the wrapped blot with protein side up in an X-ray film cassette. Place a sheet of X-ray autoradiography film on the top of the membrane. Close the cassette for 15 sec to 1 min. Remove the film for development. Add additional films if needed for longer or shorter exposures.