Bradford DC protein assay (Bio-Rad, USA) was used to measure the concentration of protein lysates. Subsequently, 20–40 μg of protein was resolved on a 12% Bis-Tris polyacrylamide gel, electrotransferred onto nitrocellulose membranes (GE Healthcare, Piscataway, USA) and blocked with 5% nonfat-milk. Protein blots were immunostained overnight with primary antibodies at 4°C and for 1 hour with secondary antibodies at room temperature. Amersham ECL Western Blotting Detection Reagents (GE Healthcare) were used to visualize the protein signal.
Bradford dc protein assay
Manufactured by Bio-Rad
Sourced in United States
The Bradford DC protein assay is a colorimetric assay for the quantitation of protein concentration. It is based on the Bradford dye-binding method and uses a detergent-compatible formulation for the determination of protein levels in the presence of interfering substances.
Automatically generated - may contain errors
Lab products found in correlation
Precast gel, by Bio-Rad (3 mentions)
Epiquik whole cell extraction kit, by AmyJet Scientific (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Horseradish peroxidase, by Merck Group (2 mentions)
Gapdh, by Merck Group (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Pvdf filter membranes, by Cytiva (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
4 to 20 mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (1 mentions)
Snail, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence detection kit, by Cytiva (1 mentions)
Skim milk, by BD (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Catalase, by Cell Signaling Technology (1 mentions)
11 protocols using bradford dc protein assay
1
Western Blot Protein Quantification
2
Hippocampal NF-κB Protein Extraction and Analysis
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Protein was extracted from the hippocampal tissue of the rats using a Nuclear and Cytoplasmic Protein Extraction kit (Pierce, Thermo Scientific, Rockford, IL, USA). The protein concentration was determined by the Bradford DC protein assay (Bio-Rad, Hercules, CA, USA). The proteins were then separated in 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane, which was then incubated with phosphate-buffered saline (PBS) containing 50 g/l skimmed milk at room temperature for 4 h. Then, the PVDF membrane was incubated with mouse anti-rat monoclonal NF-κB P65 (1:50 dilution) and mouse anti-rat monoclonal GAPDH (1:100 dilution) primary antibodies (Abcam, Cambridge, UK), respectively, at 37°C for 1 h. After washing with PBS three times, the PVDF membrane was incubated with the goat anti-mouse peroxidase-conjugated secondary antibody (Abcam) at room temperature for 1 h. Chemiluminescent detection was performed using an ECL kit (Pierce Chemical, Rockford, IL, USA).
3
Western Blot Analysis of Apoptosis Regulators
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Western blot analysis was performed as previously described (16 (link)). Briefly, total cellular protein was isolated, and the protein concentration was determined using the Bradford DC protein assay (Bio-Rad, Hercules, CA, USA). Forty micrograms of protein were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with the following primary antibodies: NIK (#4994, 1:1,000), X-linked inhibitor of apoptosis (XIAP; #14334, 1:1,000), FLICE-like inhibitory protein (FLIP; #8510, 1:1,000), B-cell lymphoma-extra large (Bcl-xL; #2764, 1:1,000) (all from Cell Signaling Technology, Inc., Boston, MA, USA) and GAPDH (1:2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and the proteins were visualized by the ECL procedure (Amersham Biosciences Corp., Piscataway, NJ, USA).
4
Western Blot Analysis of Extracellular Matrix Proteins
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Total protein from each group was extracted and the protein concentration was measured using the Bradford DC protein assay (Bio-Rad, Hercules, CA, USA). 25 μg protein from each sample was separated using SDS–polyacrylamide gel electrophoresis (10 %) and blotted onto polyvinylidene difluoride (PVDF) filter membranes (Whatman). The membranes were blocked in 5 % skim milk for 1 h, washed four times with Tris-buffered saline containing Tween 20 (TBST) at room temperature and then incubated overnight at 4 °C with diluted primary antibodies (rabbit anti-human Cthrc1/TIMP-1/MMP-2/MMP-9 antibody, 1:500, Santa Cruz). Following extensive washing with TBST, membranes were incubated with secondary antibodies at room temperature for 1 h (goat anti-rabbit IgG, 1:1000, Santa Cruz). After four 15 min washes with TBST at room temperature, immunoreactivity was visualized by enhanced chemiluminescence. GAPDH expression (Santa Cruz Biotechnology) served as an endogenous reference.
5
Nickel-Dependent Regulation of Urease in H. pylori
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Overnight H. pylori B128 parental strain, ∆hpn, ∆hpn-2 and ∆hpn ∆hpn-2 mutants grown in BBß were diluted to OD600nm 0.2 in fresh BBß either without added nickel or with 10 μM NiCl2. Aliquots were taken after 4, 8 and 24 hours culture. Bacteria were pelleted and frozen. Immuno blotting was performed on crude extracts after cell lysis in 1% SDS pH 5 according to standard protocols. Protein amounts in the crude extracts were calibrated using the Bradford DC Protein Assay (Biorad) with bovine serum albumin (BSA) as a standard. 20 μg of total extracts were separated by 12.5% SDS-PAGE using the Criterion TGX Stain Free Any kD acrylamide gels (BioRad). Gels were blotted on a nitrocellulose membrane (GE healthcare). The H. pylori UreA protein was specifically detected with an anti-ureA rabbit polyclonal antibody used at a dilution of 1:5000 [76 (link)]. Goat anti-rabbit IgG-HRP (Santa Cruz) were used as secondary antibodies and the detection was achieved with the ECL reagent (Pierce). Band intensity was quantified with a Pharos imager using Image Quant Software (Molecular Dynamics).
6
Western Blot Analysis of H. pylori Proteins
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H. pylori cells were grown to an OD600 of 0.8, harvested by centrifugation, and then washed twice in phosphate-buffered saline (PBS) prior to be disruption by sonication in a lysis buffer containing 10 mM Tris-HCl (pH 7.5), 5 mM β-mercaptoethanol, and complete protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation, and supernatants were collected as total extracts. Protein amounts in each sample were calibrated using the Bradford DC protein assay (Bio-Rad) with bovine serum albumin (BSA) as a standard. Twenty micrograms of proteins was loaded and separated on a 4-to-20% Mini-Protean TGX precast protein gel (BioRad) and subsequently electrotransferred on a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The H. pylori RhpA, RNase J, UreA, UreB, and FlaA proteins were detected with rabbit polyclonal antibodies anti-RhpA and anti-RNase J (4 (link)), anti-UreA and anti-UreB (49 (link)), and anti-FlaA (50 (link)) at the respective dilutions of 1:5,000, 1:700, 1:500, 1:5,000, and 1:2,000. Goat anti-rabbit IgG–horseradish peroxidase conjugate (Santa Cruz Biotechology) was used as secondary antibody at a 1:10,000 dilution, and the detection was achieved with chemiluminescent substrate ECL reagent (Pierce).
7
Western Blot Analysis of Protein Expression
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The EpiQuik whole cell extraction kit (AmyJet Scientific, Inc.) was used to extract total protein from the induced cells and protein concentration was measured using the Bradford DC protein assay (Bio-Rad Laboratories). Protein samples (30 µg) were separated by electrophoresis on 12% Bis-Tris polyacrylamide gel and then transferred to nitrocellulose membranes (MilliporeSigma; Merck KGaA). The membranes were blocked with 5% BSA (Wuhan Servicebio Technology Co., Ltd.) at room temperature for 1.5 h and incubated overnight at 4˚C with the following primary antibodies: SMAD 2/3 (cat. no. #3102S), ERK1/2 (cat. no. #4695T) (Cell Signalling Technology, Inc.), GSK-3 (cat. no. #PA532440; Thermo Fisher Scientific, Inc.) and SNAIL (cat. no. #ABD38; MilliporeSigma) primary antibodies at 1:1,000 dilution at 4˚C for 12 h, followed by incubation with goat anti-rabbit polyclonal HRP conjugated secondary antibodies (1:10,000 dilution in 1% milk/TBS; cat. no. #A16110; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. Protein bands were visualized using a chemiluminescence detection kit (Amersham; Cytiva) and analyzed using Tanon 5220S image analysis system (Tanon Image software version 1.0; Tanon Science and Technology Co., Ltd.). Analysis of protein expression levels was repeated thrice.
8
Western Blot Analysis of Cell Signaling
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Western blot analysis. Total protein was extracted using the EpiQuik Whole Cell Extraction kit (AmyJet Scientific, Inc., Wuhan, China) and protein concentration was measured by Bradford DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein samples (30 µg) were separated on 12% Bis-Tris polyacrylamide gel by electrophoresis and transferred to polyvinylidene difluoride membranes (EMD Millipore, Temecula, CA, USA) which was pre-blocked with 5% (w/v) skim milk (BD Biosciences) in a rotator for 2 h at room temperature. The membranes were incubated with anti-phosphorylated Smad1/2 (cat. no. 8828), Erk1/2 (cat. no. 9101) (Cell Signaling Technology, Inc., Danvers, MA, USA) and GS (cat. no. 07-817) primary antibodies (EMD Mililipore) (all at 1:1,000 dilution) at 4˚C, for 12 h, followed by incubation with a goat anti-rabbit polyclonal horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. A0208) (Beyotime Institute of Biotechnology, Shanghai, China) (1:10,000 dilution in 1% milk/TBS) for 2 h at room temperature. Protein bands were visualized using a chemiluminescence detection kit (Amersham Biosciences; GE Healthcare, Piscataway, NJ, USA). The visualized bands were analyzed by Tanon 5220S image analysis system (Tanon Image software version 1.0; Tanon Science and Technology Co., Ltd., Shanghai, China).
9
Western Blot Analysis of MMP-2, MMP-9 and Catalase
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Cells or tumors were lysed in RIPA buffer and pelleted by centrifugation. Protein concentrations were determined using a Bio-Rad DC Bradford Protein Assay (Bio-Rad Laboratories). 40 μg of protein was electrophoresed in a Bio-Rad 4–20% Precast Gel for 65 min at 120 V. The proteins were electro-transferred onto a PVDF membrane, and blocked with 5% nonfat milk in 0.1%Tween-PBS (TPBS) for 60 min. The membranes were incubated with MMP-2 and MMP-9 (1:1000, Cell Signaling Technology, 40994S and 2270S) or Catalase (1:1000, Cell Signaling Technology, 14097S) at 4 °C overnight. Membranes were washed 5 times with TPBS and incubated with secondary antibodies conjugated with horseradish peroxidase (1:50 000, Millipore,). GAPDH (1:5000, Millipore, MAB374) or Actin (1:1000, Cell Signaling Technology, 4970) was used as a loading control. After wash with TPBS, membranes were stained with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34580) and exposed to Classic Blue Autoradiography Film.
10
Western Blot Protein Analysis
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Cells were lysed in RIPA buffer and cell lysate pelleted by centrifugation. Protein concentrations were determined using a Bio-Rad DC Bradford Protein Assay (Bio-Rad Laboratories). Cell lysates (20 to 40 μg of protein) were electrophoresed in a Bio-Rad 4–20% Precast Gel. The proteins were electrotransferred onto a PVDF membrane, and blocked with 5% nonfat milk in 0.1% Tween-PBS (TPBS) for 60 min. The membranes were incubated with primary antibodies at 4 ˚C overnight. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse was used as secondary antibody. Membranes were stained with Super Signal West Pico PLUS chemiluminescent substrate (Thermo Scientific) and exposed to Classic Blue Autoradiography Film (Molecular Technologies).
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