Immunoprecipitation/protein immunoblotting method Analytical immunoprecipitation/protein immunoblotting method is a common method in the laboratory. Shanghai Jinma This chapter will specifically analyze the operation steps of immunoblotting. Please follow the steps and have any questions. . Thank you for reading.
• For preparation of whole cell lysates, please refer to the method described in Experimental Procedure 1. Immunoblot.
• Pre-clean whole cell lysate (optional) as follows: Add approximately 0.25 μg of whole cell lysate or tissue extract (see: Whole cell lysate or tissue extract table) to 0.25 μg of the corresponding control IgG (with primary antibody Host species are matched; reference control IgGs and IgG markers), and then coupled with 20 μl of the appropriate suspension agarose protein (protein A-agarose, protein G-agarose, protein A/G-agarose, or protein L-agar) Mix with sugar and incubate for 30 minutes at 4 °C.
• Centrifuge at 4 ° C and pellet at 3,000 rpm (approximately 1,000 x g) for 30 seconds. The supernatant (cell lysate) was transferred to a new microcentrifuge tube and placed at 4 ° C.
• For each 1 ml of the above pretreated cell lysate or approximately 100–1000 μg total cell lysate, add 10 μg agarose-conjugated primary antibody (equivalent to 5 μl beads) and place on a shaker at 4 ° C Incubate for 1 hour or overnight.
• If there is no agarose-conjugated primary antibody, use the following method: 1 ml of cell lysate with 1–10 μl (ie, 0.2–2 μg) primary antibody (primary antibody concentration requires titration to determine) incubation at 4° C 1-2 hours. 20 μl of matched agarose-coupled suspension (protein A-agarose, protein G-agarose, protein A/G-agarose or protein L-agarose) was added. Cap and incubate at 4 ° C for 1 hour to overnight.
• Collect the pellet after centrifugation for 30 minutes at 3,000 (equivalent to 1,000 x g) at 4 ° C. Carefully aspirate the supernatant and retain the pellet.
• Wash the pellet 2–4 times with RIPA buffer (sc-24948) (stronger) or PBS (buffer and regular solution) (slightly moderate). Repeat the centrifugation step each time.
• After the last rinse, remove the supernatant. The pellet was resuspended in 40 μl of 2x electrophoresis loading buffer (sc-24945).
• Boil the sample for 2–3 minutes. 0.75 mm thick glue is loaded 5–10 μl per 1.0 mm wide well.
• For the next electrophoresis and immunoblotting, see Immunoblotting: Experimental Procedure 1.
Note: Depending on the secondary antibody used, the primary and secondary chains of 55 kDa and 27 kDa may be detected separately. These bands can be significantly reduced if using an agarose-conjugated primary antibody or the ImmunoCruzTM IP/WB kit.
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Massking
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