Total cell proteins and cytoplasmic and mitochondrial protein fractions were extracted using the Cell Lysis Buffer for Western, IP, and Cell Mitochondria Isolation Kit, respectively (Beyotime, Haimen, China). Following separation using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, proteins were transferred onto polyvinylidene fluoride membranes, blocked with 5% BSA, and incubated with primary antibodies and corresponding HRP-conjugated secondary antibodies in sequence. Labelled membranes were visualised using an Enhanced Chemiluminescent Western Blotting Detection System (Millipore, Billerica, MA, USA). GAPDH and COX IV were used as loading controls for total/cytoplasmic and mitochondrial proteins, respectively. A Dynabeads® Coimmunoprecipitation Kit (Pierce Biotechnology, Rockford, IL, USA) was used for the co-IP analysis. Total cell, cytoplasmic, and mitochondrial proteins were incubated in anti-cofilin-1 antibodies and normal mouse IgG at 4°C overnight. Subsequently, co-IP samples were subjected to western blotting as described above, using the appropriate antibodies.
Cell lysis buffer for western ip
Manufactured by Beyotime
Sourced in China
Cell Lysis Buffer for Western & IP is a reagent used to extract and solubilize cellular proteins for subsequent analysis using Western blotting or immunoprecipitation techniques. The buffer is designed to effectively lyse cells and release the intracellular contents, preserving the native conformation and activity of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cell mitochondria isolation kit, by Beyotime (1 mentions)
Enhanced chemiluminescent western blotting detection system, by Merck Group (1 mentions)
Mouse anti gapdh monoclonal antibody, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Solarbio (1 mentions)
Western blocking buffer, by Beyotime (1 mentions)
Fusion fx system, by Vilber (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit or anti mouse igg, by Beyotime (1 mentions)
Coomassie blue fast staining, by Beyotime (1 mentions)
Bca reagent, by Beyotime (1 mentions)
4 protocols using cell lysis buffer for western ip
1
Subcellular Protein Fractionation and Co-Immunoprecipitation
2
Western Blot Analysis of CXCL13 Expression
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Protein from the sixteen samples (four groups with n = 4 per group) used in RT-qPCR was extracted from spleens with Cell Lysis Buffer for Western & IP (Beyotime, Shanghai, China) and used for expression analysis by Western blotting. Protein concentration was measured with a BCA kit (Beyotime). A 75 μg aliquot of each protein sample was loaded and then separated in SDS-PAGE 10% gels (Fdbio science, Hangzhou, China), and transferred to polyvinylidene fluoride membranes (Solarbio, Beijing, China). After blocking for 2 h at room temperature in Western Blocking Buffer (Beyotime), the membranes were incubated overnight at 4 °C with rabbit polyclonal antibody against CXCL13 (1:100 dilution; GenScript, Nanjing, China) or mouse anti-GAPDH monoclonal antibody (1:1000 dilution; Beyotime). The membranes were washed with PBS containing 0.05% Tween-20 (PBST) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (1:1000 dilution; Beyotime) at room temperature for 2 h. The resulting signals were visualized using FDbio-Dura ECL solution A and FDbio-Femto ECL solution B (Fdbio Science), collected by Fusion FX system (Vilber Lourmat, Marne-la-Vallée, France), and measured with ImageJ software [24 ].
3
Protein Extraction and Immunoprecipitation
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Proteins were extracted by Cell Lysis Buffer for Western & IP (Beyotime) supplemented with phosphatase and proteinase inhibitor. Primary antibodies against Anti-MYC-Tag mAb (DIA-AN, #2097), FLAG-Tag mAb (DIA-AN, #2064), LKB1 (27D10) Rabbit mAb (CST, #3050), Rb (D20) Rabbit mAb (CST, #9313) and p65 (CST, #8242) were added. The mixtures were incubated at 4 °C with gentle shaking overnight. Protein A/G agarose beads were added and allowed to incubate for another 2 h. The proteins were precipitated through centrifugation and dissolved in SDS sample buffer. Samples were then fractionated by 10% SDS-PAGE gel. Coomassie Blue Fast Staining (Beyotime, #P0017) was performed according to the manufacturer’s protocols.
4
Protein Lysate Preparation and Analysis
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Cells were lysed in cell lysis buffer for Western & IP (Beyotime Biotechnology) supplemented with a protease inhibitor PMSF at 4 °C for 30 min. Xenografted tumour tissues were homogenized in cell lysis buffer for Western. Protein concentrations of the lysates were measured using the BCA reagent (Beyotime Biotechnology). Equal amounts of protein were run on 10% SDS-PAGE gels, then transferred to nitrocellulose membranes (Pall), and probed with the indicated primary antibodies. Anti-rabbit and anti-mouse (1:5000) HRP-conjugated secondary antibodies were used. Blots were detected by chemiluminescence with the enhanced chemiluminescence detection reagents (PIERCE). For co-immunoprecipitation, cell lysates were incubated overnight at 4 °C with eEF2K (Santa Cruz, sc-393,366) or PKM2 (Santa Cruz, sc-365,684) antibody, then conjugated to protein A/G agarose beads while rocking. Immunoprecipitates were washed with washing buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton-X), re-suspended in 2 × loading buffer, and resolved by SDS-PAGE followed by immunoblotting analysis. The immunoprecipitates with eEF2K antibody were also subjected to protein MS analysis. Blots were quantified using Image J software and expressed by graphs.
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