To validate the expression of exosomal protein markers, serum exosomes and Huh7 cell lysates were lysed in RIPA lysis buffer (Thermo Fisher Scientific, Inc.) containing the Halt protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). Total protein concentration was quantified using the bicinchoninic acid assay method (Thermo Fisher Scientific, Inc.); equal amounts (10 µg) of protein sample were separated with 10% gel, and then transferred onto polyvinylidene difluoride membranes (MilliporeSigma). The membranes were blocked with 5% non-fat milk (in Tris-buffered saline and 0.1% Tween-20) for 1 h at room temperature, and then incubated with the following primary antibodies: Mouse anti-Alix (1:1,000; cat. no. sc53538; Santa Cruz Biotechnology, Inc.), mouse anti-CD81 (1:250; cat. no. 10630D; Invitrogen; Thermo Fisher Scientific, Inc.), rabbit anti-CD9 (1:2,000; cat. no. ab92726; Abcam) and mouse anti-BiP/GRP78 (1:1,000; cat. no. 610979; BD Biosciences). The resulting immune complexes were then probed using secondary horseradish peroxidase-conjugated anti-rabbit (cat. no. BR170-6515; Bio-Rad Laboratories, Inc.) or anti-mouse (cat. no. BR170-6516; Bio-Rad Laboratories, Inc.) antibodies. Luminescence was observed using the ChemiDoc™ Imaging System (Bio-Rad Laboratories, Inc.).
Bicinchoninic acid assay method
Manufactured by Thermo Fisher Scientific
Sourced in United States
The bicinchoninic acid (BCA) assay method is a colorimetric detection and quantification technique used to measure the total protein concentration in a sample. It relies on the reduction of Cu2+ to Cu+ by proteins in an alkaline medium, and the subsequent chelation of the Cu+ ions with bicinchoninic acid to produce a purple-colored complex that absorbs light at 562 nm. The intensity of the color is proportional to the protein concentration in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Mouse anti cd81, by Santa Cruz Biotechnology (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ab99697, by Abcam (1 mentions)
Np 40, by Solarbio (1 mentions)
Ab6728, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Keygen Biotech (1 mentions)
6 protocols using bicinchoninic acid assay method
1
Exosomal Protein Marker Validation
2
Isolation and Quantification of Mouse Microparticles
3
Western Blot Analysis of DIMT1 Protein
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Protein collection and western blot analysis were performed as previously described (25 (link)). Cells and tissue samples were lysed on ice for 30 min using RIPA lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate; Beijing Solarbio Science & Technology Co., Ltd.] before centrifuging at 4°C, 17,000 × g for 30 min. Proteins were quantified using the bicinchoninic acid assay method (Thermo Fisher Scientific, Inc.), and equivalent protein (25 µg) was loaded to each lane and separated on 10% gels using SDS-PAGE. Following this, proteins were transferred to polyvinylidene fluoride membrane and blocked with 5% skimmed milk solution at room temperature for 1 h. Subsequently, membranes were incubated with mouse anti-human primary antibodies against DIMT1 (cat. no. ab69434; Abcam) and rabbit anti-human GAPDH (cat. no. ab97627; Abcam) at 4°C overnight. After this, membranes were incubated with the rat anti-mouse (cat. no. ab6728; Abcam) or mouse anti-rabbit (cat. no. ab99697; Abcam) secondary antibody at room temperature for 1 h. Finally, protein bands were visualized using a chemiluminescence kit (cat. no. AR21PN003; AccuRef Scientific) and qualified using ImageJ software (version 1.49; National Institutes of Health).
4
Measuring Myocardial Glutathione Levels
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Reduced glutathione in the myocardium was measured by Ellman's method [25 (link)]. Briefly, an equal amount of heart tissue was homogenized in 10% (w/v) of ice-cold 0.05 M potassium phosphate buffer (pH 7.4). The resultant homogenate was centrifuged at 15,800 g for 30 minutes at 4°C. To deproteinize, 0.5 ml of the above supernatant was mixed with 0.5 ml of 5% trichloro acetic acid (TCA) and centrifuged at 2,300 g for 10 minutes. The deproteinized 0.5 ml sample is mixed with 0.25 ml of dithio-nitro-benzoic-acid (DTNB) and 1.5 ml of 0.3 M disodium hydrogen phosphate. Finally, the optical density of the mixture was measured at 412 nm. The readout of the sample was normalized by total protein present as measured by the bicinchoninic acid assay method (Thermo Scientific).
5
Lung Injury Assessment in Rats
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In another set of experiments, the lung wet-to-dry (W/D) weight ratio and protein level in BALF were determined for the animals in each group 6 or 24 h after cold-warm-cycles. For lung W/D ratio, the upper lobe of right lung of rat was excised and weighed followed by drying at 80 °C for 72 h34 (link), 35 (link). The ratio of the wet lung weight to the dry lung weight was calculated. For determination of protein concentration of BALF, the rats were inserted with a plastic cannula into the trachea and douched with 2 mL sterile saline (pH 7.4) with withdrawing for three times. BALF samples were centrifuged (1580 g, 4 °C) for 15 min and the supernatants were collected35 (link), 36 (link). The concentration of protein in the supernatants of BALF was quantitated by bicinchoninic acid assay method (Thermo Scientific, Fremont, CA, USA).
6
Western Blot Analysis of Autophagy Markers
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As described by Zhong, et al [34 (link)]. Radio immunoprecipitation (RIPA) buffer (Keygen Biotech) and phenylmethylsulfonyl fluoride were mixed at a 100:1 ratio. The protein from each group of cultured cells was extracted according to the instructions. After centrifugation at 1000 × g for 20 min, the supernatant was collected, and the bicinchoninic acid assay method was utilized for quantification of the total protein (Thermo Fisher Scientific, USA). An equal amount of protein (30 μg) was loaded in each lane. The concentrated gel was electrophoresed at 80 V for 40 min, and the separation gel was electrophoresed at 100 V for 2 h. Approximately 200 mA of current was used to transfer the polyvinylidene fluoride (PVDF) membranes (Millipore, USA), followed by the blocking step (5% skimmed dry milk). Membranes were incubated with anti-P62 (1:1000), Beclin1 (1:1000), LC3 I/II (1:1000), and β-actin (1: 5000) primary antibodies overnight at 4°C. The next day, add corresponding secondary antibodies were incubated for 2 h, and the luminescent solution was exposed and developed (Thermo Fisher Scientific). Protein expression levels were quantified with ImageJ software and normalized to the loading control β-actin.
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