Protein samples were prepared in ice-cold RIPA buffer (Tris-HCl 50 mM, NaCl 150 mM, Triton X-100 1%, vol/vol, sodium deoxycholate 1%, wt/vol, and SDS 0.1%, wt/vol, pH 7.5) supplemented with complete proteinase-inhibitor cocktail tablets (Roche Diagnostic, Shanghai, China). Protein estimation was performed by spectrophotometry with a Bio-Rad DC Protein Assay Kit using BSA as a standard and a Bio-Rad Model 680 Plate Reader. A quantity of 30 μg of total protein was separated on 8–15% SDS-PAGE and transferred to nitrocellulose membrane (Millipore). Membranes were then incubated with different primary antibodies, as indicated in Supplementary Table 2 , in blocking solution overnight at 4 °C. Final signals were detected using an ECL kit (Amersham Biosciences, Shanghai, China).
Complete proteinase inhibitor cocktail tablet
Manufactured by Roche
Sourced in Germany
Complete proteinase-inhibitor cocktail tablets are a laboratory product that helps to inhibit the activity of proteolytic enzymes, also known as proteases. These tablets contain a mixture of compounds that work together to prevent the degradation of proteins during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Ecl kit, by Cytiva (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Model 680 plate reader, by Bio-Rad (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Anti col1, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Psa elisa kit, by Abcam (1 mentions)
Human fibrinogen, by Merck Group (1 mentions)
7 protocols using complete proteinase inhibitor cocktail tablet
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Testicular Proteins
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Tissues were homogenized in ice-cold RIPA buffer (Tris-HCl 50 mM, NaCl 150 mM, Triton X-100 1% vol/vol, sodium deoxycholate 1% wt/vol, and SDS 0.1% wt/vol pH 7.5) supplemented with complete proteinase-inhibitor cocktail tablets (Roche Diagnostic, Mannheim, Germany). For western blot analysis, 30 μg mouse testicular protein was separated on 8–10% SDS/PAGE and transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). Membranes were then incubated with primary antibodies including anti-Ddr2 (R&D Systerms, dilution 1:1000), anti-β-actin (Santa Cruz biotechnology, dilution 1:2000) and anti-COL1 (Abcam, dilution 1:1000) in blocking solution overnight at 4°C. After washing with PBS containing 0.05% Tween-20 (PBST), the membranes were incubated with peroxidase conjugated goat anti-rabbit IgG or goat anti-mouse IgG (diluted at 1:10000) for 60 min. The bands were finally detected by using an ECL kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions.
3
Quantifying VEGF in Tumor Tissues
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VEGF levels were estimated in plasma, tumor tissue homogenates by ELISA method. Tumors were homogenized in RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100) containing proteinase inhibitors (Complete Proteinase Inhibitor Cocktail Tablets, Roche, Indianapolis. IN). Tissue debris was pelleted and the resulting supernatant was used in subsequent analysis. Total protein concentration was determined with a BCA protein assay (Pierce, Rockford, IL). VEGF concentrations were determined according to the manufacturer's instructions. A minimum of 3 tumors per group were analyzed in duplicate [30] (link).
4
Determination of Cell Surface Protein Levels
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For determination of relative cell surface protein levels, cells were treated with Sulfo-NHS-LC-Biotin (Pierce; 0.25 mg/ml PBS supplemented with 1 mM MgCl2 and 0.1 mM CaCl2), quenched with 100 mM glycine in DMEM and lysed in lysis buffer supplemented with Complete Proteinase Inhibitor Cocktail Tablets (Roche). Biotinylated proteins were immunoprecipitated using NeutrAvidin beads (Thermo Scientific) and analyzed by Western blotting. For detailed information see (Braune et al., 2014 (link); Preuße et al., 2015 (link)).
5
Androgen Depletion and PSA Expression
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After androgen depletion for 3 days, the cells were plated onto a 24-well plate at 2 × 10 5 cells/well in media containing 10% FBS-CS and cultured overnight, followed by R1881 incubation. After 2 days of incubation, the cells were lysed with ice-chilled cell lysis buffer (Cell Signaling) supplemented with complete proteinaseinhibitor cocktail tablets (Roche Diagnostics). The final PSA content in the supernatant was measured using PSA ELISA kit (Abcam). To further confirm the effect of hormone ablation condition during the genesis of LNCaP-R, we monitored the expression level of PSA, as described above, at different time points following the establishment of LNCaP-R. As for the latter, intracellular AR levels were quantified using AR ELISA assay (Active Motif, Shanghai, China) at different time points following the establishment of LNCaP-R.
6
Quantifying FAK Phosphorylation Dynamics
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Cells were plated on pFN-coated glass coverslips and spun in the spinning disk device, then lysed in RIPA buffer (50 mM Tris, pH 7.4; 1% NP-40; 0.5% Na-Deoxycolate; 0.1% SDS; 2 mM EDTA) supplemented with proteinase inhibitors (Complete Proteinase Inhibitor Cocktail tablet, Roche), phosphatase inhibitors (Protease Inhibitors Cocktail 2 Aqueous Solution and Cocktail 3, Sigma-Aldrich), 1 mM Na3VO4 and 5 mM NaF for 10 min on ice, and sonicated for 1 min. The protein concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific) assay and 30–50 μg of protein were separated by SDS-PAGE gel, transferred to nitrocellulose membranes and hybridized with specific antibodies. Western-blots were analyzed with ImageJ and the levels of phospho-Tyr397FAK or phospho-Tyr861FAK were referred to the total FAK content.
7
Quantification of Platelet Fibronectin Levels
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Platelets were isolated from Fn1+/+and Fn1syn/syn heparinized blood as described above. About 5 × 106 platelets were lysed with 0,1% Triton in TBS with proteinase inhibitors (Complete Proteinase Inhibitor Cocktail tablet, Roche) during 10 min on ice. After centrifugation at 13,000 rpm, the supernatant was run in an 8% SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes and incubated with anti-FN antibodies. To quantify the plasma content of FN and fibrinogen, 2 μl of plasma were loaded onto the 8% SDS-PAGE. As a reference, we used pure human pFN (Millipore) and human fibrinogen (Sigma-Aldrich). Western-blots were analyzed with ImageJ. To know the FN levels in platelets derived from the different mouse strains the FN levels were related to their vinculin contents.
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