The ALDH-high and ALDH-low cells recovered from FACS were centrifuged for 5 minutes at 270g, washed once with phosphate-buffered saline solution (PBS), and lysed in Laemmli buffer (Bio-Rad, Hercules, CA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitro-cellulose membranes. Membranes were blocked in 5% nonfat milk dissolved in 1× Tris-buffered saline (TBS), 0.1% Tween-20 (Sigma-Aldrich). Primary antibodies were dissolved in 4% bovine serum albumin (Sigma-Aldrich) in 1× TBS, 0.1% Tween-20. Secondary antibodies were prepared in 5% nonfat milk in 1× TBS, 0.1% Tween-20. Membranes were developed using Pierce ECL Chemiluminescent (Thermo Fisher Scientific, Inc, Rockford, IL). Protein quantification was performed by densitometric analysis using ImageJ Software (National Institutes of Health, Bethesda, MD). Expression is presented as sPLA2 relative to GAPDH.
Pierce ecl chemiluminescent
Manufactured by Thermo Fisher Scientific
The Pierce ECL Chemiluminescent is a laboratory equipment used for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent substrate to generate a light signal that can be captured and measured, providing a sensitive and reliable method for protein visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Tween 20, by Merck Group (2 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Laemelli buffer, by Bio-Rad (1 mentions)
Ab77256, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Sab3500023, by Merck Group (1 mentions)
Species specific horseradish peroxidase conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Pierce protease inhibitor mini tablet, by Thermo Fisher Scientific (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using pierce ecl chemiluminescent
1
Western Blot Analysis of ALDH Cells
2
TNF-α-Induced NF-κB Activation Assay
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Cells were plated on 6-well plates at a density of 3 × 106 in full media for 24 hours, then serum-starved (0.5% FBS) for 24 hours, then treated. Cells were stimulated with or without TNF-α (20 ng/mL) for 4 hours, then lysed with Laemelli buffer under reducing conditions. Time trials with TNF-α identified sustained changes in NF-κB activation at this time point (data not shown). Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis techniques and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk in 1 × PBS, 0.1% Tween-20 (Sigma-Aldrich). Primary antibodies were dissolved in 5% bovine serum albumin (Sigma-Aldrich), 1 × PBS, 0.1% Tween-20. Secondary antibodies were prepared in 5% nonfat milk in 1 × PBS, 0.1% Tween-20. The protein of interest was developed using Pierce ECL Chemiluminescent (Thermo Fisher Scientific, Inc, Rockford, Ill). Densitometric analysis was performed using ImageJ Software (National Institutes of Health, Bethesda, Md).
3
Western Blot Protein Analysis Protocol
4
Protein Extraction and Western Blotting
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Right kidneys of DO mice and LBR-null mice were homogenized in T-PER Tissue Protein Extraction Reagent (78510, Thermo Fisher Scientific) with Pierce Protease Inhibitor Mini Tablets, EDTA-Free (A32953, Thermo Fisher Scientific). The tissue lysates were then centrifuged at 20,000g for 15 min, and the supernatant was collected, subjected to SDS–polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (GE Healthcare). Membranes were blocked with 3% bovine serum albumin (BSA) in tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 1 hour. Primary antibodies diluted in TBST were treated and incubated at 4°C overnight with the following conditions: rabbit anti-H3K9me2 (1:500; 07-441, Sigma-Aldrich), goat anti-HP1α (1:500; ab77256, Abcam), rabbit anti-H3K9me3 (1:1000; 49-1008, Thermo Fisher Scientific), and chicken anti–α-tubulin (1:3000; SAB3500023, Sigma-Aldrich). After washing with TBST, the membrane was incubated with species-specific horseradish peroxidase–conjugated secondary antibodies (1:10,000; Thermo Fisher Scientific) at room temperature for 1 hour. The signals were developed with the Pierce ECL chemiluminescent (Thermo Fisher Scientific) for 1 min.
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