Pierce radioimmunoprecipitation assay ripa buffer

Samples were lysed on ice in Pierce radioimmunoprecipitation assay (RIPA) buffer (Thermo, 89900) containing complete EDTA-free protease inhibitor cocktail (Sigma/Roche, 11836170001) and debris was pelleted. Protein content was determined using Pierce Micro bicinchoninic acid (BCA) Protein Assay kit (Thermo, 23235). Samples were prepared in 4x loading dye (Bio-Rad Laboratories, Hercules, CA), boiled at 95°C for 5 min, and loaded in 4%-15% gradient Mini-Protean TGX Stain-Free precast gels (Bio-Rad). Gels were run at 175V and transferred to a 0.2-μm-pore-size nitrocellulose membrane by the semidry transfer method. Blots were blocked in 1% casein buffer/Tris-buffered saline (TBS) for 3 hours at 4°C. Primary antibodies were diluted in 1% casein and incubated overnight at 4°C. Blots were washed three times with TBS with 0.01% Tween 20 (TBST) and incubated with secondary antibody diluted in TBST for 1 hour at room temperature. Horseradish peroxidase secondary antibodies were diluted 1:10,000. Blots were washed three times with TBST and then incubated with ClarityMax according to manufacturer’s recommendation (Bio-Rad, 1075062) for HRP secondary antibodies. Blots were imaged on a ChemiDoc MP imaging system.

Neutrophils were lysed in Pierce radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) containing protease inhibitors (cOmplete Mini; Roche Diagnostics, Indianapolis, IN) and sodium orthovanadate (New England BioLabs, Ipswich, MA). Proteins were resolved by SDS-PAGE under reduced conditions and transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in Tris-buffered saline–1% Tween (TBS-T)–5% nonfat milk. Total Syk protein was detected with rabbit MAb D3Z1E anti-Syk (Cell Signaling, Danvers, MA) (1:1,000). The blots were subsequently reacted with mouse MAb AC-15 anti-β-actin (Sigma, St, Louis, MO) (1:200,000) to verify equivalent loading levels.

Cells were washed with phosphate-buffered saline (PBS) and then lysed by sonication in Pierce radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) containing cOmplete protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Aliquots of the cell lysates were mixed with sodium dodecyl sulfate (SDS) sample buffer for 5 min on ice. Polypeptides in the samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 5% to 20% gradient SDS-PAGE gels (Fujifilm Wako Pure Chemical, Osaka, Japan) and electroblotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blocking with SuperBlock (Tris-buffered saline) blocking buffer (Thermo Fisher Scientific), the membranes were incubated overnight at 4°C with primary antibodies diluted in Can Get Signal immunoreaction enhancer solution 1 (Toyobo, Osaka, Japan). The membranes were then washed three times with PBS containing 0.05% Tween 20 (PBS-T) and incubated with horseradish peroxidase-conjugated secondary antibody diluted in Can Get Signal immunoreaction enhancer solution 2 (Toyobo) for 2 h at room temperature. After washing, the membranes were treated with SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific), and the chemiluminescent signals on the membranes were detected using a ChemiDoc Touch imaging system (Bio-Rad Laboratories, Hercules, CA).