Mutant dprA::HAΩkan, ΔNter-dprA::HAΩkan and dprAΔCter::HAΩkan bacteria were grown to an A650nm of 0.4–0.6 in TGY2X and 20 mL of culture was centrifuged. The cell pellets were resuspended in 150 μL of SSC 1X buffer and the cells disrupted as described previously (Bouthier de la Tour et al., 2009 (link)). After centrifugation, the protein concentration was measured (using the Bio-Rad protein assay dye reagent kit), and 10 μg of proteins were subjected to electrophoresis through a 12% Glycine SDS polyacrylamide gel. The proteins were transferred onto a PVDF (polyvinylidene difluoride) membrane. The membrane was blocked with TBS containing 5% powdered milk and 0.05% Tween 20 before being incubated overnight at 4°C with a 1:5000 dilution of polyclonal rabbit anti-HA antibodies (Life Technologies) in TBS containing 3% powdered milk, 0.05% Tween 20. After extensive washes in TBS-0.05% Tween 20, the membrane was incubated with anti-rabbit IgG alkaline phosphatase conjugate (Promega) used as secondary antibody and revealed by a colorimetric reaction.
Protein assay dye reagent kit
Manufactured by Bio-Rad
Sourced in Spain
The Protein Assay Dye Reagent kit is a laboratory product designed to measure the concentration of proteins in a sample. The kit contains a dye reagent that changes color when it binds to proteins, allowing for quantification of the protein content using a spectrophotometer or similar instrument.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg alkaline phosphatase conjugate, by Promega (1 mentions)
Immune blot pvdf membrane, by Bio-Rad (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Stat5a, by Santa Cruz Biotechnology (1 mentions)
Drosha, by Cell Signaling Technology (1 mentions)
Phenylacetic acid, by Merck Group (1 mentions)
5 protocols using protein assay dye reagent kit
1
Western Blot Analysis of DprA Mutants
2
Immunoprecipitation of VASP Protein
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Cells were collected, lysed using western and IP lysis buffer supplemented with protease inhibitor cocktail for 30 min on ice, and centrifuged at 12,000 × g for 15 min. After protein quantification using a Bio-Rad Protein Assay Dye Reagent kit, clarified lysates were incubated with anti-VASP mouse antibody (10 μg/ml) overnight at 4°C and magnetic protein A/G beads for 4 hat 4°C. Normal mouse immunoglobulin (IgG) was used as a negative IP control. The beads were washed at least five times with western and IP lysis buffer, and proteins were eluted by boiling in 1× loading buffer at 100°C for 5 min and then used for western blotting.
3
Western Blot Analysis of SMAD3 Protein
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Tissue samples were collected after the mice were sacrificed. Proteins were extracted from liver with the T-PER tissue protein extraction reagent (Thermo Scientific). Protein quantification was measured by the protein assay dye reagent kit (Bio-RAD). Samples were loaded at equal amount and were electrophoresed on 10% SDS-PAGE gels and transferred to immune-blot PVDF membrane (Bio-Rad). After blocking with 5% nonfat milk in 1 × TBST buffer (10 mM Tris-Cl [pH 7.5], 150 mM NaCl, 0.1% Tween 20), membranes were incubated at room temperature for 1 hour with a monoclonal antibody specific to the SMAD3 (1:1000 dilution, Sigma-Aldrich) or a monoclonal antibody specific to β-actin (1:1000 dilution, Sigma-Aldrich). Membrane was then washed with 1 × TBST buffer (10 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.1% Tween 20) and incubated with 1:2000 dilution horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) at room temperature for 1 hour. Proteins were detected using the ECL system (Fisher-Scientific) and exposure to X-Ray film (Phenix).
4
Protein Expression Analysis in MV4-11 Cells
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Total protein extracts from MV4-11 cell lines were harvested using RIPA lysis buffer with protease inhibitors, and protein concentration was determined using a Bio-Rad Protein Assay Dye Reagent kit. SDS-denatured protein was separated via gel electrophoresis and transferred onto a nitrocellulose membrane. Protein was detected via overnight antibody staining with the following antibodies: STAT5A (Santa Cruz L-20), Drosha (Cell Signaling D28B1), Ago2 (Cell Signaling C34C6), p53 (Santa Cruz FL-393), and Actin (Sigma A5441).
5
Immobilization of Penicillin G Acylase on Glyoxyl-Agarose
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PGA was purchased from Merck (Madrid, Spain) as an aqueous solution (with a mean value of 86 ± 8 mg of protein per mL and 3.4 U/mg). Phenylacetic acid, 6-nitro-3-(phenyl acetamido) benzoic acid (NIPAB), ethylenediamine (EDA), glycine, ethanolamine, cysteine, glucose, aspartic and β-mercaptoethanol were also acquired from Merck. Sepharose® 4BCL was procured from ABT (Madrid, Spain). Divinyl-sulfone (DVS) was supplied from Thermo Fisher Scientific (Madrid, Spain). The Protein Assay Dye Reagent kit was purchased from Bio-Rad (Alcobendas, Spain). All other reagents were of analytical grade. Glyoxyl-PGA biocatalyst (with an enzyme loading of 2.5 mg/g) was used as reference and prepared as previously described [82 (link)]. This biocatalyst is described as among the most stable in the literature [52 (link),53 (link),54 (link)]. For this purpose, PGA was diluted in 50 mM sodium carbonate containing 100 mM Phenylacetic acid/30% (v/v) glycerol with the pH adjusted at pH 10.05. Then, glyoxyl-agarose beads were added under gently stirring. After 3 h, solid borohydride to reach a concentration of 1 mg/mL was added, and after 30 min, the biocatalyst was vacuum filtered and washed with 100 mM sodium acetate at pH 5 and with an excess of distilled water using a sintered filter. The immobilization yield was 100% and the expressed activity was 80% [52 (link),53 (link),54 (link)].
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