Protein lysates were obtained by homogenization in RIPA lyses buffer [0.1% sodium dodecylsulfate (SDS), 0,5% deoxycholate, 1% Nonidet, 100 mmol/L NaCl, 10 mmol/L Tris–HCl (pH 7.4), 0.5 mmol/L dithiotritol, and 0.5% phenylmethyl sulfonyl fluoride, protease inhibitor cocktail (Hoffmann-La Roche)] and clarification by centrifugation at 14,000 rpm for 15 min a 4 °C. Protein samples containing comparable amounts of proteins, estimated by a modified Bradford assay (Bio-Rad), were subjected to western blot and immune-complexes were detected with the enhanced chemiluminescence kit ECL plus, by Thermo Fisher Scientific (Rockford, IL) using the ChemiDoc (Bio-Rad). Each experiment was done in triplicate.
Modified bradford assay
Manufactured by Bio-Rad
Sourced in United States
The Modified Bradford assay is a colorimetric protein quantification method. It is used to determine the concentration of proteins in a sample by measuring the absorbance of the sample at a specific wavelength.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (24 mentions)
Protease inhibitor cocktail, by Roche (7 mentions)
Pvdf membrane, by GE Healthcare (6 mentions)
Ecl plus western blotting detection system, by GE Healthcare (5 mentions)
Rabbit anti mouse igg, by Bio-Rad (4 mentions)
Goat anti rabbit igg, by Bio-Rad (4 mentions)
Chemidoc, by Bio-Rad (3 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti p44 42 mapk, by Cell Signaling Technology (2 mentions)
Anti phospho p44 42 mapk, by Cell Signaling Technology (2 mentions)
Anti egfr, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Typhoon 8600, by Cytiva (2 mentions)
D4y7e, by Cell Signaling Technology (2 mentions)
Ecl prime chemiluminescent developers, by GE Healthcare (2 mentions)
T5168, by Merck Group (2 mentions)
Amersham ecl, by GE Healthcare (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Cell Signaling Technology (2 mentions)
Ecl prime, by Cytiva (2 mentions)
50 protocols using modified bradford assay
1
Protein Lysate Extraction and Western Blot
2
Western Blot Analysis of Tumor Samples
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Following treatment, cancer cells were lysed with Tween-20 lysis buffer (50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 0.1% Tween-20, 10% glycerol, 2.5 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonylfluoride, and 10 μg/mL of leupeptin and aprotinin) and protein lysates containing comparable amounts of proteins, estimated by a modified Bradford assay (Bio-Rad), were subjected to western blot. Immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus, by Thermo Fisher Scientific (Rockford, IL). Tumor samples harvested from mice were cut into 25 mm3 pieces and stored in RNA later until protein extraction for western blot analysis. Protein lysates were obtained by homogenization in RIPA lyses buffer (0.1% sodium dodecylsulfate (SDS), 0,5% deoxycholate, 1%Nonidet, 100mmol/L NaCl, 10 mmol/L Tris–HCl (pH 7.4), 0.5 mmol/L dithiotritol, and 0.5% phenylmethyl sulfonyl fluoride, protease inhibitor cocktail (Hoffmann-La Roche)) and clarification by centrifugation at 14,000 rpm for 10 minutes a 4°C.
3
Expression of Fibronectin and Laminin in MeT-5A Cells
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The expression levels of fibronectin 1 (FN1) and laminin, gamma 1 (LAMC1) in MeT-5A with/without TEX incubation were investigated by western blotting. The antibodies for FN1 (Cat. No. HPA027066) and LAMC1 (Cat. No. HPA001909) were purchased from Sigma Life Science (MO, USA). The antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology (CA, USA). The cells were harvested in M-PER lysis buffer (Pierce, Rockford, IL, USA) supplemented with protease inhibitors (Pierce, Rockford, IL, USA). Protein concentration was measured by a modified Bradford assay (Bio-Rad, Hercules, CA, USA). Cell lysates containing 20 μg of total protein were separated by SDS-PAGE and then transferred onto PVDF membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were then probed with the indicated antibodies, and proteins were detected by an ECL Plus Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA).
4
Protein Expression Analysis by Western Blot
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Cells were cultured as described above. Proteins were extracted and lysates were quantified by modified Bradford assay (Bio Rad, Mississauga, ON) and run through SDS-PAGE. Western-blots were performed by incubating a primary antibody against Galectin 8, RHOT1, USP4, TBC1D25, PIDD, or β-actin where appropriate overnight at 4°C. HRP-conjugated secondary antibodies were used to achieve detection with a chemiluminescent system.
5
Cell Lysis and Protein Quantification
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Cells were lysed with lysis buffer containing 1% (v/v) Triton X-100 and 1% (v/v) IGEPAL in PBS, supplemented with phosphatase (Sigma-Aldrich) and protease inhibitor cocktails (Roche, Mannheim, Germany) at 4 °C. Modified Bradford Assay (Bio-Rad, Hercules, CA, USA) was used to quantify total protein content of the samples.
6
Multiplex Analysis of Signaling Proteins
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Protein lysates containing equal amount of proteins, measured by a modified Bradford assay (BIORAD, Hercules, CA), were subjected to direct Western Blot (WB). Immuno-complexes were dectected with the enhanced chemiluminescence kit (ECL plus, Thermo Fisher Scientific, Rockford, IL). We used the following antibodies from Cell Signalling (Beverly, MA): anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-HER2, anti-phospho-HER2 (Tyr1248), anti-HER3, anti-phospho-HER3 (Tyr1289), anti-IGF1R-beta, anti-phospho-IGF1R-beta (Tyr1135), anti-p44/42 MAPK, anti-phospho-p44/42MAPK, anti-AKT, anti-phospho-AKT (Ser 473), anti-AXL, anti-c-MET, anti-S6 ribosomal protein, anti-phospho-S6 ribosomal protein, anti-4EBP1, anti-phospho-4EBP1, anti-vimentin, anti-E-cadherin, anti-Snail. Anti-α-tubulin (internal loading control) was from Sigma (Sigma-Aldrich, St. Louis, MO). The following secondary antibodies from Biorad were used: goat anti-rabbit IgG and rabbit anti-mouse IgG. Each experiment was done in triplicate.
7
Western Blot Analysis of SDHD Protein
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Cells were lysed for 15 min at 4°C using RIPA buffer (1% NP-40 in 150 mM NaCl, 50 mM Tris [pH 7.5], 2 mM EDTA) containing phosphatase and protease inhibitors. Proteins were quantified using a modified Bradford assay (Biorad). Protein samples (50 μg) were separated in 10% SDS/PAGE gels and electroblotted to Hybond ECL membrane (Amersham Biosciences). SDHD (polyclonal, rabbit, 1:200, Santa Cruz Biotechnology) was used. Secondary antibody was conjugated with peroxidase (Santa Cruz Biotechnology) and visualized by the ECL detection solution. Membrane was re-stained with a goat polyclonal anti-actin (Santa Cruz Biotechnology) for loading protein control. XTC1, a hurthle cell thyroid cell line, was used as positive control.
8
Protein Expression Analysis in Myocardial Tissue
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Western blot was performed as described in our previous publications [21 (link)]. In brief, the myocardial tissues were lysed with ice-cold RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor cocktail (Sigma-Aldrich, MO, USA). After protein concentration measurement by the modified Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Then, they were probed with antibodies against cleaved caspase-3, gp91phox, iNOS, p-eNOS, eNOS, Notch1 ICD, PCNA, Hes1, Pten, p-Akt, Akt, and β-actin (1 : 1000, Cell Signaling Technology, MA, USA) overnight (4°C) followed by incubation with HRP-conjugated secondary antibodies (1 : 5000 Zhongshan Biotechnology, Beijing, China) for 1 h (room temperature). The SuperSignal ECL kit (Thermo Fisher Scientific, Rockville, MD, USA) was employed to detect the antigen-antibody complexes. The bands were quantified and analyzed using an image analyzer Quantity One System (Bio-Rad, CA, USA). The results were expressed as density values normalized to β-actin.
9
Protein Isolation and Western Blot Analysis
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Total protein was isolated and quantified using a modified Bradford assay (Bio‐Rad). Western blot analyses were performed using the following primary antibodies: mouse anti‐COASY (Santa Cruz sc‐393812, 1/1,000), rat anti‐D1 dopamine receptor (Sigma D2944, 1/1,000), mouse anti‐TFR1 (ThermoFisher 13‐6800, 1/3,000), rabbit anti‐pyruvate dehydrogenase (Cell Signaling 3205, 1/3,000), rabbit anti‐VDAC (Cell Signaling 4661, 1/5,000), mouse anti‐MSH6 (BD 610918, 1/10,000), and mouse anti‐β actin (ThermoFisher AM4302, 1/10,000). Most of the primary antibody binding was visualized with the traditional method using a HRP‐conjugated secondary antibody (Jackson ImmunoResearch, 1/10,000) and ECL substrate (SuperSignal™ West Pico PLUS, ThermoFisher). However, anti‐DRD1 and its loading control (anti‐MSH6) were visualized using a biotinylated secondary antibody and Alexa Fluor™ 488‐conjugated streptavidin (ThermoFisher, 1/4,000). The fluorescent Western blot signal was captured using iBright™ FL1000 (ThermoFisher).
10
Protein Lysate Preparation and Western Blot Analysis
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Protein lysates were prepared according to standard procedures. Briefly, cells were harvested in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1.5 mM MgCl2, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 10 μg of aprotinin/ml, 10 μg of leupeptin/ml) and clarified by centrifugation at 10,000 × g. Protein concentration was estimated with a modified Bradford assay (Bio-Rad) and lysates were submitted to Western blot. Membranes were probed with the above mentioned antibodies. Immune complexes were revealed by an enhanced chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech). Signal intensity was quantitated using a Phosphorimager (Typhoon 8600, Amersham Pharmacia Biotech) interfaced with the Image Quant software.
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