Thymocyte lysates were prepared using RIPA buffer (300 mM NaCl, 2% octylphenoxypolyethoxyethanol (IGEPAL CA-630; Sigma-Aldrich), 1% deoxycholic acid, 0.2% SDS, 100 mM Tris-HCl pH 8.0) containing protease inhibitors (Roche, Basel, Switzerland) and platelet lysates with NP40 lysis buffer (1% octylphenoxypolyethoxyethanol, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) containing protease inhibitors. Proteins were separated on NuPAGE Bis-Tris gels (Life Technologies, Carlsbad, CA, USA) according to manufacturer's instructions. Blots were probed with: anti-Mcl-1 (clone 19C4-15; WEHI mAb facility), anti-human Bcl-2 (clone Bcl-2-100;46 (link) WEHI mAb facility), anti-Bcl-2 (clone 7; BD Biosciences), anti-Bim (polyclonal; Enzo Lifesciences, Farmingdale, NY, USA), anti-Bcl-xL (polyclonal; BD Biosciences), anti-Bak (polyclonal; Sigma-Aldrich) and anti-β-actin (clone AC-74; Sigma-Aldrich).
Anti bim
Manufactured by Enzo Life Sciences
Sourced in United States
Anti-BIM is a monoclonal antibody that specifically binds to the BIM (BCL2-interacting mediator of cell death) protein. BIM is a pro-apoptotic member of the BCL2 family that plays a crucial role in the regulation of apoptosis, or programmed cell death. Anti-BIM can be used to detect and quantify BIM expression in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Trans blot turbo system, by Bio-Rad (3 mentions)
Anti nur77, by BioLegend (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Any kd sds page gels, by Bio-Rad (2 mentions)
Anti bcl 2, by BD (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Anti bak, by Merck Group (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti bcl xl, by BD (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Studio lite software, by LI COR (1 mentions)
Anti fasn, by Cell Signaling Technology (1 mentions)
Irdye anti rabbit or anti mouse secondary antibodies, by LI COR (1 mentions)
Anti mcl 1, by Cell Signaling Technology (1 mentions)
Anti fasl, by BD (1 mentions)
4 protocols using anti bim
1
Western Blot Analysis of Apoptosis Regulators
2
Immunoblotting Protocol for Protein Expression Analysis
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For general immunoblotting, cells were lysed in 1% Nonidet P-40 (NP-40) lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF) + complete protease inhibitors (Roche) for 30 min on ice. Lysates were cleared by centrifugation, boiled in 2x sample buffer (Laemmli buffer + 50 μM 2-βME) and separated on 4–20% SDS-PAGE gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes for 10 min using the TransBlot Turbo system (Bio-Rad) and subsequently blocked with Odyssey blocking buffer (LI-COR). Blots were probed with the following Abs: anti-FASN (Cell Signaling Technology #3180), anti-MCL-1 (Cell Signaling Technology #94296), anti-BCL-2 (Cell Signaling Technology #4223), anti-FASL (BD Pharminogen #556372), anti-BIM (Enzo Life Sciences), anti-NUR77 (Biolegend, clone 1E10A15) and anti-β-actin (Sigma-Aldrich). Blots were incubated with IRDye anti-Rabbit or anti-Mouse secondary antibodies (LI-COR) and imaged using the Odyssey CLx instrument. Band intensity quantification was conducted with StudioLite Software (LI-COR).
3
Activated CD8+ T Cell Protein Analysis
4
Autophagic Protein Regulation in T Cells
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Expanded effector T cells (1×106 per time point) deprived of IL-2 for 0–3 days were washed in cold PBS, and lysed in 1% Nonidet P-40 (NP-40) lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF) containing complete protease inhibitors (Roche Diagnostics Corp., Indianapolis, IN, USA) for 30 min on ice. For LC3 blotting, cells were lysed in 10 mM Tris-Cl, pH 6.8, 69 mM NaCl, 0.5 mM EGTA, 0.5% Triton X-100, 5% glycerol containing complete protease inhibitors (Roche Diagnostics Corp.) for 30 min on ice. Cleared lysates were boiled in 2x reducing sample buffer and resolved on Any kD SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were transferred to nitrocellulose or PVDF (for LC3) on a Trans-Blot Turbo system (Bio-Rad), blocked with 2% Tropix I-Block (Thermo Fisher Scientific) or 6% milk (Bio-Rad) in TBS/0.1% Tween, and probed with the following Abs: anti-BIM (Enzo Life Sciences Inc); anti-PUMA (Cell Signaling Technology); anti-Bcl-xL, anti-Mcl-1, anti-Bcl-2 (BD Biosciences); anti-LC3B and anti-β-actin (Sigma-Aldrich). Bound Abs were detected using HRP-conjugated secondary Abs (Southern Biotech, Birmingham, AL USA; Agilent Technologies) and ECL (Thermo Fisher Scientific). Densitometry analysis was performed using ImageJ software (www.imagej.net ). Full blots are included as Supplementary Information .
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