Sorted purified NK cells (purity>97%) from WT: IRE1NK mixed BM chimeras day 1.5 PI were collected directly into 20% TCA (trichloroacetic acid) buffer. After sorting, the concentration of TCA was adjusted to 10% before incubation on ice for 30 min to precipitate proteins. The homogenates were centrifuged at 13, 000 r.p.m. for 10 min at 4°C, and the supernatant was carefully removed. The pellets with concentrated proteins were washed twice with acetone, and then solubilized in solubilization buffer (containing 9M Urea, 2% Triton X-100, and 1% DTT) mixed with LDS buffer (Invitrogen). The samples were heated at 70°C for 10min and protein concentrations determined using a BCA protein assay kit (Thermo Fisher Scientific). Equivalent amounts of protein for WT and KO cells from the same BM chimeras were separated by SDS–PAGE (Bis-Tris gel, Invitrogen) and transferred onto PVDF membranes following the standard protocol. Anti-cMyc (D84C12, Cell Signaling Technology), anti-β-Actin (Cell Signaling Technology) and goat anti-rabbit secondary antibodies conjugated wiih HRP (Thermo Fisher Scientific) were used. SuperSignal West Pico and Femto chemiluminescent substrates (Thermo Fisher Scientific) were used to image blots in a ChemiDoc™ MP Imaging System instrument (BioRad). Densitometric quantification was performed using Image J software (NIH).
Anti cmyc d84c12
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-cMyc (D84C12) is a rabbit monoclonal antibody that recognizes the c-Myc protein. It is designed for use in applications such as western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Lds buffer, by Thermo Fisher Scientific (2 mentions)
Femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit secondary antibodies conjugated wiih hrp, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system instrument, by Bio-Rad (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (1 mentions)
Immobilon chemiluminescent substrate, by Merck Group (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Anti β tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti glutaminase, by Abcam (1 mentions)
Goat anti rabbit horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Anti asct2, by Cell Signaling Technology (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Ba1056, by Boster Bio (1 mentions)
5 protocols using anti cmyc d84c12
1
Protein Extraction and Western Blot Analysis
2
Western Blot Protein Detection Protocol
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Dissected tissue samples were homogenized in RIPA lysis buffer containing 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM NaF; 1% NP-40; 0.25% sodium deoxycholate; 1 mM sodium orthovanadate; 1 mM EDTA and a protein protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Lysates containing 10–40 µg of protein were separated by SDS-PAGE and transferred to Hybond ECL Nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature. The membranes were then probed with the following antibodies: rabbit polyclonal anti-Id2 (sc-489; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit monoclonal anti-Id2 (D39E8, #3431; Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-Mxd1/Mad1 (sc-222; Santa Cruz Biotechnology), rabbit monoclonal anti-c-Myc (D84C12, #5605; Cell Signaling Technology) and mouse monoclonal anti-β-actin (Sigma-Aldrich, Saint Louis, MO, USA) antibodies. After incubation with horseradish peroxidase-conjugated secondary antibodies, bound proteins were detected by incubation with an Immobilon chemiluminescent substrate (Millipore, Billerica, MA, USA).
3
Western Blot Analysis of Metabolic Proteins
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Protein was isolated from cells using lysis buffer (20 mM Tris pH 8, 200 mM NaCl, 1 μm EDTA pH 8 0.5% Np40 and 10% glycerol). Lysates were standardized for protein content and 20 μg of total protein was separated by a gradient 4-20% SDS-polyacrylamide gel (Mini-PROTEAN TGX, Bio-Rad, CA, USA) electrophoresis and transferred to PVDF membranes using the iBlot system (Invitrogen, ThermoFisher Scientific, Grand Island, NY, USA). The membranes were blocked and primary antibody was diluted 1:1000 in 3% BSA in TBS containing 0.01% Tween 20 (TBST) and incubated overnight at 4°C. The following day, blots were washed in TBST buffer and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako, Carpinteria, CA, USA) for 1 hr at room temperature. After washing in TBST buffer (three times, 10 min per wash) the immunoreactive proteins were visualized using ECL detection reagent (Amersham, GE Healthcare Biosciences, Pittsburgh, PA, USA). The antibodies used were anti-ASCT2 (V501, Cell Signaling, Beverly, MA, USA) and anti-c-Myc (D84C12, Cell Signaling, Beverly, MA, USA), anti-glutaminase (Abcam, Cambridge, MA, USA) and anti-β-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA).
4
Protein Expression Analysis in Skin Tissue
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The expression of β‐catenin, c‐Myc and cyclin D1 was examined by western blotting. After trituration, protein extraction and degeneration, the skin tissue samples were separated by SDS‐PAGE and transferred onto PVDF membranes. Membranes were placed in a blocking solution and incubated with the corresponding primary antibodies (anti‐β‐catenin (D10A8), anti‐c‐Myc (D84C12) and anti‐cyclin D1 (92G2); Cell Signaling Technology) at 4°C overnight. The next day, the membranes were incubated with secondary antibodies (BA1056; Boster, China) for 1 h at room temperature. Finally, the membranes were incubated with the exposure liquid and the protein bands were detected using a Bio‐Rad detection system (Hercules).
5
Protein Extraction and Western Blot Analysis
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