Cell lysates from cultured bladder cells and human bladder tissues were prepared as previously described [13 (link)]. The protein levels were determined by the Bradford procedure (Bio-Rad, Hercules, CA). Western blots were performed as indicated in previous protocols [26 (link)]. Antibodies against human UPIII and α-tubulin were used as primary antibodies and horseradish peroxidase-labeled antibodies against mouse/rabbit IgG were used as secondary antibodies. The signals were developed with an enhanced chemiluminescence technique (Amersham Biosciences).
Bradford procedure
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The Bradford procedure is a colorimetric assay used to measure the concentration of protein in a solution. It involves the addition of an acidic dye, Coomassie Brilliant Blue G-250, to the protein sample. The dye binds to the protein, causing a shift in the dye's absorption spectrum, which can be measured spectrophotometrically. The absorbance of the resulting blue-colored solution is proportional to the protein concentration in the sample.
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Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence technique, by Cytiva (1 mentions)
Lysis buffer, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit or anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Tbs t, by Merck Group (1 mentions)
Rabbit anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Dc assay, by Bio-Rad (1 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Anti c met, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 7, by Cell Signaling Technology (1 mentions)
Anti phosphorylated egfr, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti phosphorylated akt, by Cell Signaling Technology (1 mentions)
Anti pi3k, by Cell Signaling Technology (1 mentions)
Anti egfr, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
9 protocols using bradford procedure
1
Protein Quantification and Immunoblotting of Bladder Cells
2
Comprehensive Protein Expression Analysis
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For detection of pThr308Akt, Akt, FOXO1, interleukin (IL)-1β, (IL)-4, IL-13, IL-17A, phosphorylated (p) insulin receptor (p-IR), pSer636-IRS1, IRS1, pThr183/Tyr185-JNK, JNK, pThr180/Tyr182-p38MAPK, p38MAPK, monocyte chemoattractant protein (MCP)-1, pSer2448-mTOR, mTOR, pSer536-nuclear factor kappa B (pSer536-NFkB), NFkB, pSer380/Thr382/383-PTEN, pSer727 signal transducer and activator of transcription 3 (pSer727STAT3) and STAT3, thirty mg of inguinal fat and liver were homogenized on ice in 400 μL of lysis buffer (Merck). The lysates were frozen during 16 h at −80 °C. Later, samples were centrifuged at 12,000× g for 5 min at 4 °C and the supernatants kept at −80 °C until assayed [12 (link)]. Protein levels were measured by the Bradford procedure (Bio-Rad Laboratories).
3
Quantification of Collagen and HtrA1 Proteins
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Scaffolds with differentiated cells or undifferentiated monolayer cells were washed with cold PBS, ground and total proteins were extracted by RIPA lysis buffer containing protease inhibitors. Protein concentration was evaluated by the Bradford procedure (Bio-Rad SA, Rosedale, New Zealand). Proteins lysates (20 μg) were separated on 10% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Non-specific binding sites of membranes were blocked with 10% non-fat milk powder in Tris-buffered saline containing 0.1% Tween (TBST, Sigma-Aldrich) for 1 h. Then, the blots were incubated overnight at 4 °C with anti-type I collagen (Novotec, 1:3000, Lyon, France), or anti-type II collagen (Novotec, 1:1500), or anti-type X collagen (Sigma, 1:5000), or rabbit anti-HtrA1 (Millipore, 1:2500). The membranes were also reacted with rabbit anti-GAPDH (Santa Cruz Biotechnology, Inc., 1:5000, Dallas, TX, USA) to verify equal loading. The next day, membranes were washed with TBST and, then incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch, 1:5000, West Grove, PA, USA) for 1 h at room temperature with 3% milk in TBST. Bound antibodies were detected using a chemiluminescence assay (Western Lightning® Plus-ECL, PerkinElmer, Inc., Waltham, MA, USA) and exposed to X-ray film for visualization.
4
Protein Extraction and Western Blot Analysis
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At the end of treatments, cell were lysed with HEPES lysis buffer (50 mM HEPES [pH 7.6], 150 mM NaCl, 20 mM sodium pyrophosphate, 10 mM NaF, 20 mM beta-glycerophosphate, 1% Triton, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mM Na3VO4, and 1 mM PMSF). The protein concentrations were analyzed using the Bradford procedure (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard for all samples. The samples (40 µg of protein) were boiled in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and resolved on SDS-PAGE gels, transferred onto polyvinylidene difluoride membrane, and analyzed by Western blotting with appropriate antibodies.
5
Protein Extraction and Western Blotting
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First, the cells were harvested, washed and lysed in ice-cold western blot buffer (100 mM Tris-HCL, pH 8, 150 mM NaCl, 1% NP-40, 1% protease inhibitor cocktail) for 30 min on ice. These lysates were then centrifuged at 14 000 r.p.m. for 15 min at 4 °C and the supernatant assayed for protein concentrations by the Bradford procedure (Bio-Rad, Sundbyberg, Sweden), followed by western blotting using standard procedures.
6
Metabolic Profiling of Parasitic Samples
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The levels of the metabolites in the biological samples are expressed as intensity/2 x 106 promastigotes (parasite extracts) or intensity/2 μl medium (media samples). To ensure validity of comparisons between samples, the protein content of the parasite pellets collected were determined according to the Bradford procedure (BioRAD), using bovine serum albumin as the protein standard.
7
Protein Extraction from Frozen Chondrocytes
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Sponges containing chondrocytes were frozen and ground and the subsequent extraction of the total proteins was performed using RIPA-lysis buffer supplemented with a cocktail of protease inhibitors (phenylmethylsulfonyl fluoride, pepstatin A, aprotinin and leupeptin) and the phosphatase inhibitor sodium orthovanadate for 45 min at 4 °C as previously described [62 ]. Protein concentration was determined according to the Bradford procedure (Bio-Rad, Hercules, CA, USA).
8
Protein Isolation and Co-Immunoprecipitation
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Cells were lysed in RIPA buffer (10 mM Tris-HCl pH 7.6, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P40, 0.5% sodium deoxycholate) in the presence of protease and phosphatase inhibitor cocktail (Sigma, Aldrich). Solubilized proteins were obtained in the supernatant after centrifugation at 13,000 rpm for 5 min. Protein concentrations were estimated by the Bradford procedure (Bio-Rad) for all samples except those containing SDS where the DC assay (Bio-Rad) was used. Co-immunoprecipitations were performed on pre-cleared solubilized proteins or supernatants using APP (22C11) antibody, pan-Trk antibody (B3) and non-relevant monoclonal mouse IgG antibody as control. WB were performed using rabbit APP-CT (1:2000), APP-NT (1:2000), rabbit monoclonal TrkA-CT (1:2000) and rabbit TrkA-NT (1:1000) antibodies. Densitometry of the WB was analyzed with the Image J software (NIH) and normalized to corresponding reference protein β-actin.
9
Protein Quantification and Western Blot Analysis
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After treatment, proteins were isolated with RIPA buffer (Cell Signaling Technology, USA), and then quantified using the Bradford procedure (Bio-Rad, USA). Approximately 50 μg of total proteins were then separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, USA). Membranes were incubated in 5% skim milk for 1 h at room temperature and then incubated with the primary antibodies (anti-EGFR, anti-phosphorylated EGFR, anti-PI3K, anti-phosphorylated PI3K, anti-AKT, anti-phosphorylated AKT, anti-c-met, anti-cleaved caspase-9, anti-cleaved caspase-7, anti-cleaved caspase-3 and anti-β-actin, all of them purchased from Cell Signaling Technology) overnight at 4°C. A fluorescent goat anti-rabbit (Cell Signaling Technology) secondary antibody was used and the protein bands were then detected with an enhanced chemilu-minescence detection kit (Pierce, USA). The level of β-actin was used to normalize the protein levels.
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