The MCF-7 and HCT-116 cells were lysed in ice-cold NP40 lysis buffer containing protease cocktail-inhibitor (Sigma-Aldrich). The protein concentrations were calculated using the Bradford method (Bio-Rad). Then, 30 μg protein were loaded and separated by 12% SDS-PAGE and then blotted onto a nitrocellulose membrane (Bio-Rad). The membrane was then blocked with 5% skimmed milk powder, then washed with Tris-buffered saline, 0.1% Tween 20 (TBST), and incubated at 4 °C overnight with primary IgG-unlabeled antibodies, anti-survivin (EPR2675) (ab134170); anti-CDK1 (A17) (ab18) and anti–cyclinB (Y106) (ab32053), purchased from Abcam, Cambridge, MA, USA. Secondary antibodies (antimouse and antirabbit; Cell Signaling Technology) were reacted for one hour with the membrane at 1:1000 dilutions. Chemiluminescence (Thermo Fisher Scientific) was used to detect the bands and Image Lab software (ChemiDoc Touch Gel and Western blot imaging system; Bio-Rad) was used to quantify the band-density; β-actin was used as a normalization control.
Chemidoc touch gel and western blot imaging system
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc™ Touch Gel and Western Blot Imaging System is a versatile imaging platform designed for capturing high-quality images of gels and blots. It utilizes a sensitive CCD camera and a range of illumination sources to provide accurate and reproducible results for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Bradford method, by Bio-Rad (3 mentions)
Ecl kit, by Thermo Fisher Scientific (3 mentions)
Secondary antibodies antimouse and antirabbit, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab201119, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ab156034, by Abcam (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Phospho akt s473, by ABclonal (1 mentions)
Phospho mtor s2448, by ABclonal (1 mentions)
Anti jmjd2b, by Abcam (1 mentions)
Protease inhibitor cocktail tablet, by Merck Group (1 mentions)
Ripa buffer, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Anti mouse and anti rabbit secondary antibodies, by Cell Signaling Technology (1 mentions)
10 protocols using chemidoc touch gel and western blot imaging system
1
Western Blot Analysis of Cell Signaling
2
Western Blot Analysis of FTO Protein
3
MMP-1 Protein Expression in PdLFs
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To investigate the protein expression of MMP-1 in PdLFs, the cells were treated with 1 µM, 10 µM or 100 µM l -lactic acid compared to 1 µM PMA for 2 and 6 days and the western blot was performed according to Hamoudi et al.75 (link). The cell pellets were lysed in RIPA buffer (ab156034, Abcam, UK) supplemented with 1:10 protease and phosphatase inhibitor cocktail-EDTA free (ab201119, Abcam, UK). The protein lysates were quantified using the Pierce BCA protein assay kit (Thermo-Scientific, USA). 10 µg Protein samples were used for MMP-1 detection using MMP-1 (54,376) rabbit monoclonal antibody and normalized with β-actin (13E5) rabbit monoclonal antibody according to the manufacturer’s instructions. The blots were visualized using the Clarity Western ECL Substrate (Bio-Rad, USA) in the ChemiDoc Touch Gel and Western Blot Imaging System (Bio-Rad, USA). Image Lab Software (V 6.1.0) was used to detect and quantify the protein bands.
4
Investigating Signaling Pathways in DAOY and HDMBO3 Cells
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To assess treatment effects on target proteins, 2.5 × 105 DAOY wild type or 2.5 × 105 HDMBO3 wild type cells were seeded per well in 6 well plates in complete growth medium and incubated overnight at 37 °C. Medium was replaced with serum-free medium. After overnight incubation at 37 °C, cells were treated with bFGF (100 ng/mL), SAG (100 nM), BGJ398 (1 µM), or in combination for 24–48 h. Treatments were changed every 24 h. Cells were lysed using RIPA buffer and processed for immunoblot (IB) with antibodies against GLI1 (1:1000, Cell Signaling Technologies, Danvers, MA, USA), ERK1/2 (1:1000, Cell Signaling Technologies), and phospho-ERK1/2 (1:1000, Cell Signaling Technologies, Danvers, MA, USA). Loading was normalized using GAPDH (1:1000, Cell Signaling Technologies) or Tubulin (1:1000, Sigma) detected on the same membrane. HRP-linked secondary antibodies (1:5000, Cell Signaling Technologies) were used to detect the primary antibodies. Chemiluminescence detection was performed using ChemiDoc Touch Gel and Western Blot imaging system (BioRad, Hercules, CA, USA) and FujiFilm LAS 3000 (Bucher Biotech, Basel Switzerland) Integrated density of immuno-reactive bands was quantified using ImageJ open source image processing program (https://imagej.net/ ).
5
Western Blot Analysis of Carnosic Acid Effects
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An ice-cold NP40 lysis buffer containing protease cocktail-inhibitor (Sigma-Aldrich) was used to lyse both the carnosic acid-treated and untreated AGS cells. Thirty micrograms of protein was separated using 12% SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Skimmed milk was used to block the membrane, followed by washing with tris-buffered saline with 0.1% tween solution (TBST). The membrane was incubated with primary IgG-unlabeled antibodies of AKT1 (#A17909), Phospho-AKT-S473 (#AP0637), mTOR (#A2445), and Phospho-mTOR-S2448 (#AP0115), purchased from ABclonal technology (Woburn, MA, USA), and survivin (EPR2675) (ab134170) purchased from Abcam, Cambridge, MA, USA, at 4 °C overnight. Later, the membrane was incubated with secondary antibodies (anti-mouse and anti-rabbit; Cell Signalling Technology) at 1:1000 dilutions for one hour. Chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) and Image Lab software (ChemiDoc Touch Gel and Western blot imaging system; Bio-Rad) were used to detect the bands and quantify band-density, respectively. β-actin was used as a normalization control.
6
Histone Modification Analysis by Western Blot
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Cells were lysed in ice-cold RIPA buffer (Abcam) containing protease inhibitor cocktail tablets (Sigma). Whole cell lysate protein concentrations were quantified using the standard Bradford method (Bio-Rad). Lysate aliquots containing 30-50 μg of protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was then blocked by 5% skimmed milk powder for 1 h at room temperature, washed with TBST, and reacted with primary immunoglobulin G (IgG) unlabeled primary antibodies; anti-JMJD2B (Abcam), various modified and total histones (Cell signaling), β-actin (Sigma), at 1 : 1000 dilution overnight at 4°C. The secondary (anti-mouse and anti-rabbit) antibodies (Cell Signaling) were then reacted with the membrane at 1 : 1000 dilution for 1 h at room temperature. Chemiluminescence was detected using the ECL kit (Thermo Scientific Pierce). Protein band quantification was carried out using the Bio-Rad Image Lab software (ChemiDoc™ Touch Gel and Western Blot Imaging System; Bio-Rad). β-Actin was used as a normalization control.
7
Angiogenesis Protein Profiling in Breast Cancer Cells
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Fifty-five angiogenesis-related proteins were measured in MCF-7 and MDA-MB-231 cells using the Human Angiogenesis (Pro-VM formation mediators) Array Kit (Cat. No. ARY007; R&D Systems, Minneapolis, MN, USA). Whole-cell lysate protein concentrations were quantified using the standard Bradford method. Four nitrocellulose membranes, each containing 55 different capture antibodies, were blocked by Array Buffer 6 for 1 h at room temperature. Lysate aliquots containing 300 μg of protein were prepared with Array Buffer 4 and 20 μl of Detection Antibody Cocktail. Samples were then loaded onto the membrane overnight at 2°C–8°C. Chemiluminescence was detected by streptavidin–horseradish peroxidase (HRP) methods using the dilution factor suggested by the manufacturer. Protein dot quantification was done using the Bio-Rad Image Lab software (ChemiDoc™ Touch Gel and Western Blot Imaging System; Bio-Rad). Reference spots were used as a normalization control; values of control (untreated) samples were defined as 1.00; values of experimental samples were quantified relative to that of control.
8
Subcellular Fractionation and Western Blot Analysis
9
Quantitative Western Blot Analysis
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Cells were lysed in ice-cold NP-40 lysis buffer (1.0% NP-40, 150 mM of NaCl, 50 mM of Tris-Cl, pH 8.0) containing protease inhibitor cocktail tablets (Cat. No. S8830; Sigma, Germany). Protein concentration of cell lysate was quantified using the standard Bradford method (Cat. No. 500-0006; Bio-Rad, Hercules, CA, USA). 50 µg of lysate protein aliquots were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad, USA). 5% skimmed milk powder was used to block the membrane at room temperature for 1 h. The membrane was then washed with TBST, and incubated with the following primary antibodies (anti-PRMT-5, anti-β-catenin, anti-H4R3me2s [Symmetrical dimethylation on arginine-3 of histone H4], anti-β-actin (all from Abcam, UK), ( cyclin D1, cdk4, cdk6, caspase-3 and PARP, all from Cell Signaling Technology, USA) overnight at 4 °C. Secondary antibodies (Cell Signaling Technology, USA) were incubated with the membrane at 1:1000 dilution for 1 h at room temperature. Chemiluminescence was detected using the ECL kit (Thermo Scientific Pierce, USA). Bio-Rad Image Lab software (ChemiDoc™ Touch Gel and Western Blot Imaging System; Bio-Rad) was used to detect and quantify protein bands. Protein levels were normalized to β-actin and ratios were calculated based on the values of control (untreated) samples.
10
Western Blot Analysis of Signaling Pathways
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RIPA buffer lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer's instructions (Bio-Rad). Membranes were probed with primary antibodies against phospho-FRS2, FRS2, ERK1/2, phospho-ERK1/2, MYPT1, phosphor-MYPT1, phospho-PKC and tubulin. HRP-linked secondary antibodies (1:5000) were used to detect the primary antibodies. Chemiluminescence detection was performed using ChemiDoc Touch Gel and Western Blot imaging system (BioRad) and FujiFilm LAS 3000 (Bucher biotech) Integrated density of Immuno-reactive bands was quantified using Adobe Photoshop CS3.
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