5/16/2023 0 Comments Western blot procedure![]() (Optional: Add 10 µl of 10 mg/ml PMSF stock sc-3597) Incubate 30 minutes on ice. Further disrupt and homogenize cells by hydrodynamic shearing (21-gauge needle), dounce homogenization or sonication, taking care not to raise the temperature of the lysate.Gently resuspend cells in RIPA buffer with a pipet and incubate on ice for 30 minutes. Add 1.0 ml of ice cold RIPA buffer ( sc-24948) with freshly added Protease Inhibitors and/or Phosphatase Inhibitors.Wash the pellet with PBS at room temperature, and again collect by low-speed centrifugation.200xg) at room temperature for 5 minutes. Collect approximately 2.0 x 10 7 cells by low-speed centrifugation (e.g.For increased protein recovery, resuspend the pellet in a small volume of RIPA, centrifuge and combine supernatants. Transfer the supernatant to a new microcentrifuge tube. The supernatant fluid is the total cell lysate. Centrifuge cell lysate at 10,000xg for 10 minutes at 4° C.(Optional: Add 10 µl of 10 mg/ml PMSF ( sc-3597) stock and/or pass through a 21-gauge needle to shear the DNA.) Incubate 30–60 minutes on ice. Wash the plate once with 0.3 ml of RIPA buffer and combine with first lysate.Transfer the resulting lysate to a microcentrifuge tube. Remove adherent cells with a cell scraper. Gently rock plate for 15 minutes at 4° C. Add 0.6 ml of RIPA buffer ( sc-24948) to the monolayer cells in the plate.The following steps should be performed on ice or at 4° C using fresh, ice cold buffers. Grow cells to subconfluency in a 100 mm x 20 mm petri dish, remove culture medium and rinse cell monolayer with room temperature 1x PBS ( 10X liquid PBS: sc-24946).Follow the procedure suited to your needs. Sample preparation procedures are provided for monolayer cells, suspension cells, and tissue samples. NITROCELLULOSE, PVDF AND NYLON MEMBRANES.AUTORADIOGRAPHY FILM, TAPE AND PLASTIC WRAP.Biologically Active Proteins & Peptides.NUCLEOTIDES, PEPTIDES, PROTEINS & AMINO ACIDS.BIOLOGICALLY ACTIVE & PURIFIED PROTEINS.PROTEIN A, G-PLUS & L AGAROSE IP REAGENTS.ISOTYPE SPECIFIC MOUSE IgG BINDING PROTEINS.POLYCLONAL GOAT/RABBIT ANTI-MOUSE SECONDARY ANTIBODIES.MONOCLONAL MOUSE ANTI-RABBIT/GOAT SECONDARY ANTIBODIES.
0 Comments
Leave a Reply. |