A549 cells were seeded in 12-well plates and cultured for 24 h. Then the A549 cells were incubated with different drug formulations at the equal concentration of Pt (15 μm ) for 48 h. The P-glycoprotein (P-gp) expressions were evaluated by flow cytometry analysis and western blot assay. For flow cytometry analysis, the cells were collected and stained with PE-conjugated mouse antihuman P-gp antibody (1:50, ab93590, Abcam, UK). The amounts of fluorescent signal were quantified using flow cytometry system (LSRFortessa, BD, USA). For western blot assay, the cell lysates were separated by SDS-PAGE gel, and extracted proteins in the gel were transferred by the polyvinylidene difluoride (PVDF) membranes. Then, the membranes were incubated with primary antibodies against P-gp and GAPDH for 12 h at 4 °C and secondary antibodies for 2 h at room temperature. The binding of antibody was evaluated using ECL Prime Western Blotting Detection Reagent (GE Healthcare UK Ltd., UK) and the images were obtained by ChemiDoc XRS system.
Chemidoc xrs system
Manufactured by GE Healthcare
Sourced in United Kingdom
The ChemiDoc XRS system is a laboratory equipment designed for imaging and analysis of gel-based samples, such as Western blots and gel electrophoresis. It utilizes a charge-coupled device (CCD) camera and a series of interchangeable filters and illumination sources to capture high-quality images of chemiluminescent, fluorescent, and colorimetric samples.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime western blotting detection reagent, by GE Healthcare (4 mentions)
Ab93590, by Abcam (2 mentions)
Lsrfortessa, by BD (2 mentions)
Low fluorescence pvdf membrane, by Bio-Rad (2 mentions)
Criterion stain free 4 20 gels, by Bio-Rad (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Ab124974, by Abcam (1 mentions)
Ab187139, by Abcam (1 mentions)
Pa1 860, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Amersham ecl prime regent, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti p erk, by Santa Cruz Biotechnology (1 mentions)
Anti p p38, by Bioworld Technology (1 mentions)
Anti p jnk, by Bioworld Technology (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Ripa lysis buffer, by BioTeke (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
7 protocols using chemidoc xrs system
1
Evaluating P-glycoprotein Expression in A549 Cells
2
Western Blot Analysis of Histone Acetylation
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Example 5
Proteins were separated on Criterion Stain-Free 4-20% gels (Biorad 567-8095) at 200V for 50 min. Proteins were transferred to low fluorescence PVDF membrane (Biorad 162-0264) at 0.14 amps for 60 min. Gels and membranes were imaged with a Chemidoc XRS system (Biorad 170-8265) for quality control purposes. Membranes were processed as follows: blocked in Tris buffered saline+Tween 20 (TBST, 0.1% Tween 20) containing 5% blocker (Biorad 170-6404) overnight at 4° C. The following steps were performed at room temperature: The membrane was washed in TBST, incubated with primary antibodies in TBST containing 1% blocker (acetyl histone H3 lysine 9: EMD Millipore 06-942-S 1:4000, acetyl histone H4 lysine 12: EMD Millipore 07-595 1:4000) for 60 min, washed in TBST, incubated with secondary antibody in TBST containing 1% blocker (anti-rabbit-HRP: Cell Signaling #7074S 1:5000, anti-mouse-HRP: Cell Signaling #7076S 1:5000) for 60 min, washed in TBST, developed with ECL prime western blotting detection reagent (GE RPN2232), and visualized with a Chemidoc XRS system. Western blot images were converted from Image Lab 5.2.1 (.scn) files to 600 dpi .tif files. The images were opened in ImageJ. Images were converted to 8-bit and background was subtracted with a rolling ball radius of 50.0 pixels. Images were inverted and mean band intensity was quantified with the measurement tool.
3
Western Blot Analysis of HDAC Proteins
4
Sensitive Western Blot Analysis of Cellular Proteins
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Western blots were performed using whole-cell extracts in protein lysis buffer (20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 50 mM Na4P2O7, 30 mM NaF, 5 μM ZnCl2, 2 mM Iodoacetic acid, 1% Triton-X) with freshly added 1 mM sodium orthovanadate and protease inhibitor cocktail (Sigma-Aldrich), separated on 8% SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T). The membrane was then incubated in a 1:2,000 dilution of a primary antibody in 5% bovine serum albumin–TBS-T at room temperature for 1 h or at 4 °C for 16 h. After washing three times with TBS-T, the membrane was incubated with 1:10,000 dilution of the corresponding secondary antibody in 2.5% non-fat dry milk–TBS-T at room temperature for 2 h. Respective proteins were developed by using Amersham ECL Prime Regent (GE Healthcare Biosciences) and image were obtained by ChemiDoc XRS+System. Images have been cropped for presentation. Full-size images are presented in Supplementary Figs 7–9 .
5
Investigating Protein Expression in Brain Tissue
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Western blotting was used to investigate the protein expression of BMP9, ERK, P38 and JNK. The protein samples were extracted from brain tissues or astrocytes using RIPA lysis buffer (BioTeke Corporation, Beijing, China) and then protein levels were determined using the bichioninic acid method. A 20 µg protein sample was subjected to 12% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes. Blots were blocked with 5% non-fat milk solution for 1 h at room temperature and then incubated overnight with anti-BMP9 (cat. no. sc514211; 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-p-ERK (cat. no. BS74621; 1:2,000; Bioworld Technology, Inc., St. Louis Park, MN, USA), anti-p-P38 (cat. no. BS6381; 1:2,000; Bioworld Technology, Inc.), or anti-p-JNK (cat. no. Bs4763; 1:2,000; Bioworld Technology, Inc.) at 4°C followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (cat. no. ZB2301; 1:3,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for 1 h. The blots were visualized with the ECL system (GE Healthcare Life Sciences, Little Chalfont, UK) and the intensity of each band quantified by using the Chemi Doc XRS system.
6
Protein Extraction and Western Blot Analysis
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Whole-cell extracts were prepared by sonication in Laemmli buffer. Standard methods were used for SDS-PAGE and protein immunoblots. Pounceau S was used to determine the loading amount. Blocking of nitrocellulose membranes performed with Blocking reagent 10X (Roche) or 5% milk, 0.05 Tween 20, TBS. Antibodies were incubated in blocking solution or in 3% BSA, 0.05% Tween 20, TBS. Primary antibodies were incubated overnight at 4°C and secondary antibodies 1h at RT. Blot signal was detected with SuperSignal West Pico Plus Chemiluminescent substrate (Thermofisher) or ECL Western Blotting Detection Reagents (GE Healthcare) either in in X-OMAT LS (Kodak) or Amersham Hyperfilm ECL (GE Healthcare) chemiluminescence films or in ChemiDoc XRS system with ImageLab 6.0.1 software.
7
Evaluating P-glycoprotein Expression in A549 Cells
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