Smarca4FLAG ESCs or mouse tissues were lysed in RIPA buffer (Sigma) in the presence of Protease Inhibitor cocktail (Sigma) for 1–2 h at 4°C with constant agitation. Standard Bradford protocol was then used to determine the protein concentration in each lysate. Protein was denatured and loaded on NuPAGE Novex 3%–8% Tris-Acetate gradient gel (Invitrogen) following the manufacturer’s protocol. Following electrophoresis, protein transfer on PVDF membranes was achieved with the XCell II Blot Module (Invitrogen). FLAG detection was obtained by incubation of blocked PVDF membranes with ANTI-FLAG M2 monoclonal antibody-peroxidase conjugate (Sigma, A8592) for 30–60 min and staining with TMB Liquid Substrate solution (Sigma).
Anti flag m2 monoclonal antibody peroxidase conjugate
Manufactured by Merck Group
The ANTI-FLAG M2 monoclonal antibody-peroxidase conjugate is a laboratory reagent used for the detection and purification of FLAG-tagged proteins. The antibody is conjugated to the enzyme peroxidase, which enables the detection of the FLAG-tagged proteins through colorimetric or chemiluminescent methods.
Automatically generated - may contain errors
Lab products found in correlation
Substrat hrp immobilon western, by Merck Group (2 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Anti flag affinity gel beads, by Merck Group (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Flag m2 magnetic beads, by Merck Group (1 mentions)
Monoclonal anti flag m2, by Merck Group (1 mentions)
Ha 11, by Fortrea (1 mentions)
Ecl western blotting kit, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Skim milk, by BD (1 mentions)
6 protocols using anti flag m2 monoclonal antibody peroxidase conjugate
1
Immunodetection of FLAG-tagged Smarca4 Proteins
2
Labeling and Protein Interaction Assays
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L-[1-14C] ornithine was purchased from Moravek Biochemicals Inc. (Brea, CA). Anti-FLAG M2 monoclonal antibody peroxidase conjugate, anti-HA monoclonal antibody peroxidase conjugate, anti-FLAG affinity gel beads, Igepal CA-630, cycloheximide, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt (BS3), MG132 (Z-Leu-Leu-Leu-al) and chloroquine were obtained from Sigma Aldrich. Lipofectamine 2000 transfection reagent, Dulbecco’s Modified Eagle Medium (DMEM), glutamine, fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA). QuickChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). [1,4-14C] Putrescine (specific activity 107 mCi/mmol) was from Amersham Biosciences. Pierce ECL Plus Western Blotting Substrate was from Thermo Scientific (IL, USA). Primers were purchased from Sigma Genosys.
3
Immunoprecipitation of FLAG-tagged Proteins
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0.5 g of 3-week-old seedling tissue were ground in liquid nitrogen, resuspended in 1.5mL ice-cold IP buffer [50mM Tris pH 7.6, 150mM NaCl, 5mM MgCl2, 0.1% Nonidet P-40, 10% glycerol, 0.5 mM DTT, 1x Protease Inhibitor Mixture (Roche)], and centrifuged 2 times for 15 min at 4°C, 16 000g. 50μL M2 magnetic FLAG-beads (Sigma, M8823) were added to the supernatants and incubated for 2 hour rotating at 4°C. Beads were washed 3 times in ice-cold IP buffer for 10 min rotating at 4°C. Immunoprecipitated proteins were denatured in Laemmli buffer for 5min at 95°C. 10μL of input and bead elution were run on 10% SDS-PAGE gels, and proteins were detected by western blotting using either Anti-FLAG M2 monoclonal antibody-peroxidase conjugate (Sigma, A8592) at a dilution of 1:10000, or c-Myc rat monoclonal antibody (Chromotek, 9E1-100) at a dilution of 1:1000 followed by goat anti-rat IgG horseradish peroxidase (Abcam, ab205720) used at a dilution of 1:20000 as secondary antibody. Western blots were developed using Substrat HRP Immobilon Western (Merck Millipore, WBKLS0500).
4
Antibody Detection and Characterization
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Antibodies used in this study: monoclonal anti-FLAG M2 (Sigma-Aldrich), anti-FLAG M2 monoclonal antibody peroxidase conjugate (Sigma-Aldrich), monoclonal antibody, HA.11 (Covance), monoclonal antibody anti-HA (clone HA-7; Sigma-Aldrich), monoclonal c-Myc (Covance), and peroxidase-conjugated Affinipure anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.).
5
Protein Extraction and Western Blot
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Total proteins were extracted from leaves of 3-wk-old seedlings using 8 M urea and denatured in Laemmli buffer for 5 min at 95°C. 10–15 μl of protein extracts were run on 10% SDS–PAGE, and proteins were detected by Western blotting using Anti-FLAG M2 monoclonal antibody–peroxidase conjugate (A8592; Sigma-Aldrich) at a dilution of 1:10,000. Western blots were developed using Substrat HRP Immobilon Western (WBKLS0500; Merck Millipore).
6
SDS-PAGE and Western Blot Analysis
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All the solubilized protein was subjected to SDSpolyacrylamide gel-electrophoresis [mini-gel (10 cm × 10 cm), 1 mm thickness, consisting 3% (w/v) stacking gel (60 V) and 18% (w/v) separation gel (100 V)] ( 24), and then electro-blotted [200 mA, 90 min.; ATTO Model-AE6675 (ATTO, Tokyo, Japan)] onto an Immobilon™-P membrane [Millipore (Billerica, MA, USA) polyvinylidene difluoride (PVDF) membrane (0.45 μm), IPVH00010] (25). The filter was washed with PBS and then blocked 1hr at 4°C with PBS plus 0.1 % (v/v) Tween 20 (PBS-T) containing 5% (w/v) skim-milk (BD, Franklin Lakes, NJ, USA). The filter was washed with PBS-T and then reacted for 2 hr at room temperature with ANTI-FLAG M2 ® monoclonal antibody-peroxidase conjugate (Sigma) (× 1,500 diluted). Chemiluminescence was detected with an ECL Western blotting kit (GE Healthcare) using Scientific Imaging Film (KODAK, Rochester, NY, USA).
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