Total crude protein was extracted from frozen-ground seedlings in the extraction buffer [50 mM sodium phosphate (pH 7.0), 100 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate]. The supernatant was collected after centrifugation at 21,000 × g for 5 min. Then protein samples were separated by 8 or 12% SDS-PAGE gels (8% for TPL, and 12% for CDF1) and transferred to nitrocellulose membranes (for each sample, 5–10 μg of total protein was used). The 3×FLAG and 6xHis epitope-tagged CDF1, CDF1-ΔN, CDF1-mut and HA-tagged TPL, HA-tagged tpl-1 fusion proteins were detected using HRP conjugated anti-FLAG (Sigma) and anti-HA (Roche) antibodies. Super Signal West Pico and Femto Chemiluminescent substrate kits (Thermo Fisher Scientific) were used to detect signals. All experiments were performed at least three times with independent biological replicates.
Femto chemiluminescent substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Femto Chemiluminescent Substrate Kit is a sensitive solution for detecting low-level chemiluminescent signals in Western blotting applications. The kit provides a stable chemiluminescent substrate that produces a glow-type signal for the detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico, by Thermo Fisher Scientific (4 mentions)
Horseradish peroxidase conjugated donkey anti rabbit or antimouse igg, by Santa Cruz Biotechnology (2 mentions)
β actin, by Merck Group (2 mentions)
Anti flag, by Merck Group (1 mentions)
Anti ha, by Roche (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Pdcd10, by Abcam (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using femto chemiluminescent substrate kit
1
Protein Extraction and Detection Protocol
2
Quantitative Protein Extraction and Analysis
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Total proteins were extracted from 200,000 BxPC-3 cells following shRNA transduction, using Laemmli SDS reducing buffer (50 mM Tris-HCl pH 6.8, 2% SDS and 10% glycerol) at 4°C, boiled and quantified using a bicinchoninic acid protein assay. Equal amounts (30 µg) of extracted protein samples were resolved by 8–10% PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 3% milk at room temperature for 1 h, incubated with primary antibodies against SATB1 (cat no. ab92307; 1:1,000; Abcam, Cambridge, UK) and GAPDH (cat no. 2118; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat no. sc-2030, Santa Cruz Biotechnology, Inc.) at a dilution of 1:5,000 at room temperature for 1 h. Protein bands were visualized by enhanced chemiluminescence using the SuperSignal West Pico or Femto Chemiluminescent Substrate kits (Thermo Fisher Scientific, Inc.). Blots were semi-quantified using ImageJ software version 1.41 (National Institutes of Health, Bethesda, MD, USA) (17 (link)).
3
Western Blot Analysis of PDCD10
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Total cell lysates were prepared by harvesting cells in Laemmli S.D.S reducing buffer [50 mM Tris‐HCl (pH 6.8), 2% S.D.S, and 10% glycerol], boiled and resolved on an 8–10% polyacrylamide gel, and transferred to polyvinylidinefluoride. Antibodies against PDCD10 (Abcam, Cambridge, MA, USA), and β‐actin (Sigma‐Aldrich) were used. The blots were incubated with horseradish peroxidase‐conjugated donkey anti‐rabbit or antimouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:5000 and detected with SuperSignalWest Pico or Femto Chemiluminescent Substrate Kit (Thermo Scientific, Grand Island, NE, USA).
4
Quantitative RT-PCR and Western Blot Analysis
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Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the First Strand cDNA Synthesis Kit (Thermo Scientific). Quantitative RT-PCR (qRT-PCR) of the resulting cDNA was performed using gene-specific primers, in triplicate, on a Mx3000P system (Stratagene, La Jolla, CA, USA). The primers for qRT-PCR were listed in Table S6. Samples from whole-cell lysate or immunoprecipitated beads were subjected to SDS-PAGE and transferred on to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were incubated with indicated primary antibodies overnight at 4 C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 hour. The antibody-bound proteins were detected by SuperSignalâ West Pico or Femto Chemiluminescent Substrate Kit (Thermo Scientific, Wilmington, DE, USA) following the manufacturer's protocol.
5
Western Blot Analysis of MLH1
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Total cell lysates were prepared by harvesting cells in Laemmli SDS reducing buffer [50 mM Tris/HCl (pH 6.8), 2% SDS and 10% glycerol], boiled and resolved on an 8–10% polyacrylamide gel, and transferred to polyvinylidinefluoride. Antibodies against MLH1 (Abcam) and β-actin (Sigma–Aldrich) were used. The blots were incubated with horseradish-peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) at a dilution of 1:5000 and detected with SuperSignal West Pico or Femto Chemiluminescent Substrate Kit (Thermo Scientific).
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