Cellular extracts from EPCs were prepared according to standard protocols (Andrews and Faller, 1991 (link)). Protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Cytoplasmic extracts (50 μg) or nuclear extracts (50 μg) were separated in a 4–20% Ready Gel Tris-HCl gel (Bio-Rad Laboratories), transferred onto nitrocellulose membranes in a semi-dry transfer system. The membrane was immediately placed into blocking buffer containing 5% nonfat milk and sequentially blotted against monoclonal primary antibodies (NFκB p65, IκBα, p53, and β-actin). All antibodies were from Cell Signaling (Boston, MA, USA) except β-actin (Santa Cruz, Dallas, TX, USA). Protein levels were quantified using the image analysis software Quantity One 4.4.0 (Bio-Rad Laboratories), using β-actin as a loading control.
Ready gel tris hcl gel
Manufactured by Bio-Rad
Sourced in United States
The Ready Gel Tris-HCl gel is a pre-cast polyacrylamide gel used for protein electrophoresis. It is available in a variety of formats and concentrations to accommodate different applications. The gel provides a consistent and reliable platform for separating proteins based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (4 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Mab374, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
β actin, by Bio-Rad (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Ecl western blotting detection system, by Cytiva (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse, by Abcam (1 mentions)
Western lighting ultra chemiluminescence substrate kit, by PerkinElmer (1 mentions)
Mouse anti ha 11 clone 16b12 monoclonal, by Fortrea (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Spectramax plus 384, by Molecular Devices (1 mentions)
12 protocols using ready gel tris hcl gel
1
Western Blot Analysis of EPC Extracts
2
Quantitative Western Blot Analysis of NF-κB Pathway
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Cellular extracts from THP-1 were prepared according to standard protocol [54 (link)]. Total protein concentration was determined using the colorimetric reagent of the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). To determine specific protein contents, cytoplasmic and nuclear extracts (50 μg each) were separated in a 4–20% Ready Gel Tris-HCl gel (Bio-Rad Laboratories) and then transferred to a nitrocellulose membrane. The membranes were blocked in TTBS with 5% milk for 1 h at room temperature and incubated with primary antibodies, including anti-p65-NF-κB and IκB (both from Cell Signaling Technology, Danvers, MA, USA) and β-actin as a loading control (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. Specific bands were visualized using ECL Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, UK). Subsequently, the intensity of the bands was quantified through densitometry analysis using Image J software (version 1.52p) by measuring the integrated optical density (IOD).
3
Quantitative Western Blot Analysis of MagA-HA
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Cells were lysed in RIPA buffer. Total protein concentration was determined by DC™ Protein Assay kit (Bio-Rad). A total of 30 µg of protein was separated using 4-15% gradient SDS-PAGE (4-15% Ready Gel® Tris-HCL Gel; Bio-Rad) and transferred onto a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was probed with primary antibodies that specifically recognized HA (mouse anti-HA.11 clone 16B12 monoclonal 1:1,000; Covance) and α-tubulin (mouse anti-α-tubulin clone DM1A monoclonal; Sigma). The membrane was then incubated with secondary antibody (HRP-conjugated goat anti-mouse 1:10,000; Abcam) after thorough washing and visualized with a Western Lighting™ Ultra Chemiluminescence Substrate kit (PerkinElmer) using the ChemiDoc™ XRS+ system (Bio-Rad). The quantification of MagA-HA was performed using ImageLab™ software (Bio-Rad) and was normalized to α-tubulin.
4
Western Blot for Protein Interactions
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Western blot was performed as described previously [17 (link)]. Eluate from co-IP were run on 4–15% Ready Gel Tris-HCL gel (BioRad), and transferred onto PVDF membranes (BioRad). Membranes were incubated overnight at 4°C with primary antibody. Secondary incubation was performed the following day, and membranes visualized and bands quantified using the LiCor Odyssey imaging systemTM.
5
Protein extraction and Western blot
6
Cardiac Differentiation Protein Detection
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Western blots were performed to detect proteins characteristic of cardiac differentiation. Whole proteins were extracted from the cells using radio-immunoprecipitation assay buffer (Thermo Scientific) according to manufacturer instructions. The protein content was quantified using the Bradford method. Absorbance at 595 nm was read using a microplate reader (Spectra max 384 Plus, Molecular Devices). Protein for each sample, 60 µg, was loaded to perform electrophoresis in a precast 4–20% SDS-PAGE (Ready Gel Tris-HCl Gel, BioRad) and transferred onto a nitrocellulose membrane (Whatman) using a semi-dry method. Proteins were detected using the following primary antibodies: mouse monoclonal anti-alpha cardiac actin (Sigma, A9357, 1∶1,000), rabbit anti-cardiac troponin I–C (Santa Cruz, sc-98830, 1∶1,000), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (Millipore, MAB374, 1∶850). Incubations were performed overnight at 4°C. The secondary antibodies used were anti-Rabbit IgG, horseradish peroxidase-linked whole Ab (GE Healthcare, NA934, 1∶10,000) and anti-mouse IgG, horseradish peroxidase-linked whole Ab (GE Healthcare, NA931, 1∶10,000).
7
Western Blot Analysis of Protein Signaling
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Protein extraction was carried out as described previously.(25 (link)) Lysates were electrophoresed on 10% Ready Gel Tris-HCl Gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to Immobilon-P filters (Millipore, Billerica, MA, USA). The filters were first incubated with primary antibodies for 2 h (c-MET, p-MET, p-AKT, p-ERK1/2, and cleaved-PARP) and 1 h (α-tubulin) at room temperature and then with HRP-conjugated secondary antibodies. The following antibodies were used: c-MET, p-MET (Tyr1234/1235), p-AKT (se473), p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), cleaved-PARP (BD PharMingen, San Jose, CA, USA), α-tubulin (Sigma-Aldrich, St. Louis, MO, USA) and HRP-conjugated secondary antibody (GE Healthcare Bioscience, Little Chalfont, UK). α-tubulin was used as a loading control. The relative band intensities were determined by densitometry using NIH Image (U. S. National Institutes of Health, Bethesda, MD, USA).
8
Western Blotting of Brain Tissue
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Human brain tissue was homogenized and sonicated in Tris lysis buffer containing 7.4% EDTA, 3.8% EGTA, and 1% Triton X-100. Lystates were centrifuged at 21,000g to remove the nuclear fraction. Samples were diluted in equal amounts of RIPA and DTT containing reducing buffer (Pierce TM catalog number 39000) to a final amount of either 30 or 40 μg protein per well. Samples were run on 4–15% Ready Gel Tris-HCL gel (BioRad) and transferred onto PVDF membranes (BioRad). Membranes were incubated overnight at 4 °C with primary antibody. Secondary incubation was performed the following day and membranes visualized and bands quantified using the LiCor Odyssey imaging systemTM. Values for proteins of interest were normalized to beta actin expression for each subject.
9
SDS-PAGE Protein Quantification and Visualization
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SDS-PAGE was carried out according to the stacking gel procedure as described by Laemmli (1970) (link). Protein concentration was quantified using a Synergy H1 plate reader (Bio-Tek Instruments, Inc., USA) with the take microdrop addition. Each sample was redissolved in SDSPAGE sample buffer [62.5 mM Tris-HCl, pH 6.8; 2% (w/v) SDS; 25% (v/v) glycerol; 5% (v/v) 2-mercaptoethanol; 0.01% (w/v) bromophenol blue] and denatured at 100℃ for 5 min. Twenty micrograms of the samples were loaded on 10% Ready Gel (Tris-HCl Gel, Bio-Rad, Hercules, USA). The equipment employed was the Mini-PROTEAN® Tetra Cell (Bio-Rad). The gels were stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio- Rad). Destaining was carried out with a Coomassie Brilliant Blue R-250 Destaining Solution (Bio-Rad).
10
Pancreatic Differentiation Protein Analysis
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Western blots were performed to detect proteins characteristic of pancreatic differentiation. At the end of the differentiation period, whole proteins were extracted from the cells using RIPA Buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail (Thermo Scientific)) according to the manufacturer's instructions. The protein content was quantified using the Bradford method (Bio-rad, 500-0006). Absorbance at 595 nm was read using a Spectra max 384 Plus plate reader (Molecular Devices). Protein for each sample, 60 µg, was loaded to perform the electrophoresis in a precast 4-20% SDS-PAGE (Ready Gel Tris-HCl Gel, BioRad) and transferred onto a nitrocellulose membrane (Whatman) with a semi-dry method. The proteins were detected using the following primary antibodies: Rabbit anti-PDX-1 (Abcam, ab47267, 1:100) rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (Millipore, MAB374, 1:850). Incubations were performed overnight at 4°C. The secondary antibodies used were ECL anti-Rabbit IgG, horseradish peroxidase-linked species-specific whole Ab (GE Healthcare, NA934, 1:10000), ECL anti-mouse IgG, horseradish peroxidase-linked species-specific whole Ab (GE Healthcare, NA931, 1:10000).
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