To assess protein–protein interactions, cells were lysed with a lysis buffer for Western and IP (Beyotime, Jiangsu, China) supplemented with protease inhibitors. The supernatant of cell lysates was collected by a centrifuge at 12,000 rpm for 30 min at 4 °C, and subsequently precleared using Protein A/G Agarose beads. After incubation with the corresponding primary antibody for 2 h at 4 °C, the supernatant was used for immunoprecipitation by the addition of beads with rotation overnight at 4 °C. The following day, beads were washed five times with the NP-40 Lysis Buffer (Beyotime), and then bound proteins were eluted from the beads by boiling in an SDS loading buffer. Afterward, the immunoprecipitates were analyzed using Western blotting.
Lysis buffer for western and ip
Manufactured by Beyotime
Sourced in China
Lysis buffer for Western and IP is a solution designed for the extraction and solubilization of proteins from biological samples. It is commonly used in Western blotting and immunoprecipitation techniques to prepare cell or tissue lysates.
Automatically generated - may contain errors
Lab products found in correlation
Np 40 lysis buffer, by Beyotime (1 mentions)
Proteinase and phosphatase inhibitor, by Selleck Chemicals (1 mentions)
Anti t mapk, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Anti pmapk, by Cell Signaling Technology (1 mentions)
2 protocols using lysis buffer for western and ip
1
Protein-Protein Interaction Assay
2
Western Blot Analysis of Fly MAPK
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About 30 fly heads were collected and homogenized in lysis buffer for western and IP (Beyotime) containing 1% proteinase and phosphatase inhibitor (Selleck) for each sample. The equivalent of 2.4 fly heads was loaded in each lane for SDS-PAGE. The resolved proteins were then transferred to a nitrocellulose membrane. The membrane was then blocked for 2 hr at room temperature in 5% skim milk in TBST (Tris-buffered saline with 0.1% Tween-20) and incubated with primary antibody (anti-P-MAPK, 1:4,000, Cell Signaling; or anti-T-MAPK, 1:4,000, Cell Signaling) overnight at 4 C. After three or more washes in TBST, the membrane was incubated with HRP-conjugated secondary antibody for 1 hr (1:4,000, Cell Signaling). The signal was detected using an ECL kit (Millipore). Data analysis was performed using ImageJ software (National Institutes of Health).
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