S. aureus strains were grown at 37°C to exponential growth phase (OD600 ≈ 0.5) at 37°C. Cells were collected, washed once with 10 mM Tris-HCl (pH 8.0), and suspended in TSM buffer (20 mM Tris-HCl, 0.5 M sucrose, 10 mM MgCl2, pH 8.0) containing lysostaphin (40 μg/ml), followed by incubation at 37°C for 30 min. After centrifugation (4,600 ×g, 5 min), the pellet was suspended in ice-cold membrane buffer (10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.0) and subjected to sonication. Non-ruptured protoplasts were removed by a brief centrifugation at 4,600 ×g, and the membrane fraction was recovered after a 45 min centrifugation at 45,000 ×g (Beckman L8-55). The membranes were suspended in 10 mM Tris-HCl (pH 8.0), 2 M KCl and centrifuged for 30 min at 120,000 ×g. The supernatant was discarded, and the pellet was suspended in 10 mM Tris-HCl (pH 8.0), 5 mM EDTA. Finally, the membranes were suspended in 1× TKMG buffer (50 mM Tris-HCl, 50 mM KCl, 1 mM MgCl2, 25% glycerol, pH 8.0). The protein concentration was determined by the bicinchoninic acid assay (Bio-Rad) and immunoblotting with anti-SaeS antibodies. The membranes were stored at -80°C until used.
Bicinchoninic acid assay
Manufactured by Bio-Rad
Sourced in United States
The Bicinchoninic acid assay is a colorimetric method for the quantitative determination of total protein concentration. It is a widely used technique in various fields, including biochemistry, molecular biology, and protein research.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Phosphatase protease inhibitors cocktail, by Roche (2 mentions)
Reducing laemmli buffer, by Bio-Rad (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Luminata forte, by Merck Group (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
L8 55, by Beckman Coulter (1 mentions)
Filter paper, by Macherey-Nagel (1 mentions)
Pro q emerald 300 glycoprotein gel and blot stain kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Coomassie brilliant blue, by Bio-Rad (1 mentions)
Anti lamin a c, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions)
Anti p acc, by Cell Signaling Technology (1 mentions)
Anti acc, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescent substrate, by Merck Group (1 mentions)
27 protocols using bicinchoninic acid assay
1
Isolation and Purification of S. aureus Membranes
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S. aureus strains were grown at 37°C to exponential growth phase (OD600 ≈ 0.5) at 37°C. Cells were collected, washed once with 10 mM Tris-HCl (pH 8.0), and suspended in TSM buffer (20 mM Tris-HCl, 0.5 M sucrose, 10 mM MgCl2, pH 8.0) containing lysostaphin (40 μg/ml), followed by incubation at 37°C for 30 min. After centrifugation (4,600 ×g, 5 min), the pellet was suspended in ice-cold membrane buffer (10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.0) and subjected to sonication. Non-ruptured protoplasts were removed by a brief centrifugation at 4,600 ×g, and the membrane fraction was recovered after a 45 min centrifugation at 45,000 ×g (Beckman L8-55). The membranes were suspended in 10 mM Tris-HCl (pH 8.0), 2 M KCl and centrifuged for 30 min at 120,000 ×g. The supernatant was discarded, and the pellet was suspended in 10 mM Tris-HCl (pH 8.0), 5 mM EDTA. Finally, the membranes were suspended in 1× TKMG buffer (50 mM Tris-HCl, 50 mM KCl, 1 mM MgCl2, 25% glycerol, pH 8.0). The protein concentration was determined by the bicinchoninic acid assay (Bio-Rad) and immunoblotting with anti-SaeS antibodies. The membranes were stored at -80°C until used.
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S. aureus strains were grown at 37°C to exponential growth phase (OD600 ≈ 0.5) at 37°C. Cells were collected, washed once with 10 mM Tris-HCl (pH 8.0), and suspended in TSM buffer (20 mM Tris-HCl, 0.5 M sucrose, 10 mM MgCl2, pH 8.0) containing lysostaphin (40 μg/ml), followed by incubation at 37°C for 30 min. After centrifugation (4,600 ×g, 5 min), the pellet was suspended in ice-cold membrane buffer (10 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 8.0) and subjected to sonication. Non-ruptured protoplasts were removed by a brief centrifugation at 4,600 ×g, and the membrane fraction was recovered after a 45 min centrifugation at 45,000 ×g (Beckman L8-55). The membranes were suspended in 10 mM Tris-HCl (pH 8.0), 2 M KCl and centrifuged for 30 min at 120,000 ×g. The supernatant was discarded, and the pellet was suspended in 10 mM Tris-HCl (pH 8.0), 5 mM EDTA. Finally, the membranes were suspended in 1× TKMG buffer (50 mM Tris-HCl, 50 mM KCl, 1 mM MgCl2, 25% glycerol, pH 8.0). The protein concentration was determined by the bicinchoninic acid assay (Bio-Rad) and immunoblotting with anti-SaeS antibodies. The membranes were stored at -80°C until used.
2
Beef Protein Extraction and Analysis
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Beef was purchased at a local butcher's store, shock‐frozen with liquid nitrogen, reduced to small pieces with a mortar and stirred in PBS containing protease inhibitors (Roche Diagnostics GmbH, Rotkreuz, Switzerland) overnight at 4°C. Thereafter, the extract was centrifuged at 10 000 g for 30 min and the supernatant was filtered through filter paper (Macherey‐Nagel, Düren, Germany), lyophilized and stored at −20°C. The protein concentration was determined by bicinchoninic acid assay (Bio‐Rad Laboratories, Richmond, CA, USA). The extract (20 μg) was separated by 12% SDS‐PAGE under nonreducing conditions and stained with Coomassie brilliant blue (Bio‐Rad Laboratories). Detection of glycosylation was performed with the Pro‐Q® Emerald 300 Glycoprotein Gel and Blot Stain Kit (Thermo Fisher Scientific) according to the manufacturers’ protocol. For immunoblot experiments, the separated extract was transferred to a nitrocellulose membrane. After blocking, sera were incubated overnight at 4°C. Bound IgE was detected with 125I‐labelled anti‐human IgE antibody (Demeditec Diagnostics, Kiel‐Wellsee, Germany) and visualized by autoradiography. Buffer and sera of nonallergic donors served as negative controls.
3
Western Blot Analysis of AMPK Signaling
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Cell lysates were prepared by collecting cells in NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris, pH 8, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM NaF, 1 mM NaVO3, 1 mM phenylmethylsulfonyl fluoride) and cleared by centrifugation at 14,000 rpm. Protein concentration was determined by bicinchoninic acid assay (Bio-Rad, Hercules, CA). Lysates (10–20 μg protein/well) were resolved by SDS–PAGE and transferred to polyvinylidene fluoride membranes for immunoblotting. Blots were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (BSA/TBST) and incubated at 4°C overnight with rabbit anti-pAMPK (1:1000; Cell Signaling, Danvers, MA), anti-AMPK (1:2000; Cell Signaling), anti–peroxiredoxin 3 (1:2000; Abfrontier/Axxora, Farmingdale, NY), anti-ACC (1:1000; Cell Signaling) anti-pACC (1:1000; Cell Signaling), anti–retinoblastoma protein (pRB, 1:1000; Cell Signaling), anti–filamin A (1:1000; EMD Millipore, Billerica, MA), or anti–lamin A/C (1:1000; Cell Signaling) in 5% BSA/TBST. Blots were incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies (1:2500; EMD Millipore) for 30 min at room temperature. Enhanced chemiluminescent substrate from Millipore was used to detect HRP-conjugated secondary antibodies on x-ray film.
4
Protein Extraction and Western Blotting
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Proteins from transfected cells were extracted using RIPA buffer (Sigma) supplemented with Protease Inhibitor Cocktail (Roche). The protein concentrations of the lysed samples were measured using the bicinchoninic acid assay (Bio‐Rad). The proteins were electrophoresed on a 10% SDS‐PAGE and were then transferred to the polyvinylidene difluoride (PVDF) membranes. After incubating with 5% skimmed milk for 1 hour at room temperature, the PVDF membranes were probed with primary antibodies against TGFBR2 (Cell Signaling Technology) and β‐actin (Cell Signaling Technology) by a further overnight incubation at 4°C. Afterwards, the PVDF membranes were washed with phosphate buffered saline with Tween‐20 for 3 times × 5 mins and were then incubated with horseradish peroxidase‐labelled secondary antibodies (Cell Signaling Technology). The Western blot signals were visualized using the ELC Substrates (ThermoFisher Scientific).
5
Protein Extraction and Western Blot Analysis
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RPE and neuroretina samples were extracted with a RIPA buffer (1% Triton X-100, 100 mmol/l Tris-HCl, pH 7.5; 150 mmol/l NaCl; 1.5 mmol/l EDTA, pH 8.0; 25 mmol/l NaF; 0.5 mmol/l Na3VO4; 0.1 mmol/l phenylmethylsulfonyl fluoride; and Complete protease inhibitors [Roche, Basel, Switzerland]) and homogenized by sonication. Protein concentrations were determined using bicinchoninic acid assay (Bio-Rad Laboratories, Madrid, Spain). Equivalent amounts (20 µg) of total protein extracts were resolved by 10% SDS-PAGE and transferred to ECL nitrocellulose membranes (Hybond, Amersham Pharmacia Biotech). The membranes were incubated with a primary antibody against VAP-1 (1:1000; ab115574, Abcam, Madrid, Spain) and further incubated with a peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Madrid, Spain). Membranes were stripped and re-probed with β-actin to evaluate the lane-loading control. Proteins were visualized using the enhanced chemiluminescence detection system ECL (Advansta, CA).
6
Gelatin Zymography for MMP Activity
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The MMP activity of AF cells was investigated by analyzing the conditioned medium through gelatin zymography, as previously described (Cardoso et al., 2014 (link)). Briefly, the total protein content in the conditioned medium was determined by bicinchoninic acid assay (Bio-Rad), and 15–20 mg of protein was mixed with sample buffer (10% sodium dodecyl sulfate, 4% sucrose, and 0.03% bromophenol blue in 0.5 M Tris–HCl, pH 6.8) and separated on 10% polyacrylamide gels containing 0.1% gelatin (Sigma-Aldrich) as substrate. After electrophoresis, gels were washed twice with 2% Triton X-100. gelatin gels were subsequently incubated for 16 h at 37°C in 50 mM Tris–HCl, pH 7.5, and 10 mM CaCl2. Gels were stained with 0.1% Coomassie Brilliant Blue R-250 (Sigma-Aldrich), 50% methanol, and 10% acetic acid (Merck). The molecular weight and activity of the MMPs were estimated by densitometric analysis.
7
Western Blot Analysis of Cellular Proteins
8
Candida albicans Carriage and Killing in Saliva
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Saliva samples were collected by expectoration and placed in a 10× protease inhibitor cocktail (cocktail set III; Calbiochem/EMD, Gibbstown, NJ, USA), and saliva was centrifuged for 5 minutes at 550 × g. Baseline oral C. albicans carriage was determined by plating the supernatant fraction of spun saliva in triplicate on yeast peptone dextrose plates with antibiotics (to suppress growth of oral bacteria) and C. albicans colony enumeration after incubation at 30°C for 48 hours. Salivary C. albicans killing was determined by incubating the salivary supernatant at 37°C with 1 × 106C. albicans cells (strain CAF2-1) (1:1, v/v) for 1 hour
[21 (link)]. C. albicans cells were plated in triplicate for colony enumeration. For β-defensin 2 (BD2) assessment, the supernatant was analyzed using a BD2 enzyme-linked immunosorbent assay kit in duplicate or triplicate as volume allowed (Phoenix Pharmaceuticals, Burlingame, CA, USA). BD2 concentrations were normalized to the total protein content of centrifuged saliva, which was measured by the bicinchoninic acid assay (BioRad, Hercules, CA, USA).
[21 (link)]. C. albicans cells were plated in triplicate for colony enumeration. For β-defensin 2 (BD2) assessment, the supernatant was analyzed using a BD2 enzyme-linked immunosorbent assay kit in duplicate or triplicate as volume allowed (Phoenix Pharmaceuticals, Burlingame, CA, USA). BD2 concentrations were normalized to the total protein content of centrifuged saliva, which was measured by the bicinchoninic acid assay (BioRad, Hercules, CA, USA).
9
Overproduction and Purification of SaeS and SaeR Proteins
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The MBP-SaeS-His6 and SaeR-His6 proteins were overproduced in E.coli BL21 (DE3) harboring plasmids pMCSG19-saeS or pET28a-saeR. Overnight cultures were inoculated into fresh LB broth, and the proteins were expressed by the addition of 1 mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG) to the fresh culture. The bacterial culture was further incubated at 16°C for 16 h (MBP-SaeS-His6) or at 37°C for 6 h (SaeR-His6). The proteins were purified with Ni-column chromatography (Qiagen) by following the manufacturer’s recommendations. The purified MBP-SaeS-His6 and SaeR-His6 were dialyzed first in 1× TKM buffer (50 mM Tris-Cl, 50 mM KCl, 1 mM MgCl2, pH 8.0) and 1× TBS buffer (10 mM Tris-HCl, 138 mM NaCl, 2.7 mM KCl, pH 7.5), respectively, and then in TKM or TBS buffer containing 25% glycerol. The purified proteins were concentrated with Amicon Ultracell-30 (MW 30,000; Millipore) for MBP-SaeS-His6 or Ultracell-15 (MW 10,000; Millipore) for SaeR-His6. Protein concentration was determined by the bicinchoninic acid assay (Bio-Rad), and the purified proteins were stored at -80°C until used.
10
Western Blot Analysis of Spn Proteins
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Cultured Spn isolates were resuspended by RIPA buffer (Sigma) with proteinase inhibitor cocktail (Sigma) and lysed by the freeze-thaw method. Supernatants were collected, and the protein concentration was measured by bicinchoninic acid assay (Bio-Rad, Hercules, CA). Cell lysates (10 μg) were run on SDS-PAGE gels, and gels were electroblotted to a 0.45-μm-pore-size nitrocellulose membrane (Bio-Rad). The membrane was blocked with 3% bovine serum albumin in T-PBS (PBS containing 0.05% Tween 20) for 1 h at room temperature. The membranes were exposed to anti-NPB antibody (PR-1A4.7) (15 (link)) or anti-PspA antibody (1b2.21) for 1 h at room temperature, washed, and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (diluted 1:10,000 in PBS-T) for 1 h at room temperature. After washing, the membranes were developed with enhanced chemiluminescence (ECL) solution (Bio-Rad) and observed by using a ChemiDoc (Bio-Rad).
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