Cells were seeded into a plate and transfected using Lipofectamine 2000 (Invitrogen), as recommended by the manufacturer. The cells were harvested 48 h after transfection or stimulation. Cell samples were boiled in 1× loading buffer [0.08 mol/L Tris (pH 6.8), 2.0% SDS, 10% glycerol, 0.1 mol/L dithiothreitol, and 0.2% bromophenol blue) and subsequently separated on a 12% polyacrylamide gel. Proteins were transferred to nitrocellulose membranes, which were probed with various primary antibodies against the proteins of interest. Primary antibodies were diluted with 1% milk in phosphate-buffered saline (PBS) and incubated with primary antibodies followed by incubation with the corresponding AP-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA). Protein staining was performed using 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium obtained from Sigma (St. Louis, MO, USA).
5 bromo 4 chloro 3 indolylphosphate and nitroblue tetrazolium
Manufactured by Merck Group
Sourced in United States
5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium are a pair of chemical compounds commonly used as a colorimetric detection system in various laboratory procedures. The 5-bromo-4-chloro-3-indolylphosphate is a phosphatase substrate, while the nitroblue tetrazolium is a redox indicator. When these two compounds are used together, they can produce a colored precipitate that indicates the presence of a specific enzyme or analyte in a sample.
Automatically generated - may contain errors
2 protocols using 5 bromo 4 chloro 3 indolylphosphate and nitroblue tetrazolium
1
Western Blot Analysis Protocol
2
Screening Adult C. sinensis cDNA Library
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An adult C. sinensis cDNA expression library was constructed as previously described [17 (link)]. The cDNA library, 1.1 × 106 pfu, was mixed with Escherichia coli XL1-Blue and cultured on a LB-agar plate. The plate was overlaid with a nitrocellulose (NC) membrane (Amersham, UK), which was treated previously with 10 mM isopropyl-D-thiogalactoside (IPTG), and then incubated at 37°C for 4 h. The membrane was further incubated for 3 h with pooled sera of five clonorchiasis patients diluted 1:100 at room temperature (RT), followed by incubation with the goat anti-human IgG alkaline phosphatase-conjugated secondary antibody (Sigma, USA) diluted 1:2,000. A color signal was developed with the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Sigma, USA) solution. Positive clones were purified through secondary and tertiary screenings using the same sera. These clones were then excised in vivo to single-stranded phagemids by employing ExAssist helper phage (Stratagene, USA), and converted to double-stranded plasmids according to the manufacturer’s instructions.
The positive clones were sequenced and analyzed, and sequence homology was searched in NCBI using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi ).
The positive clones were sequenced and analyzed, and sequence homology was searched in NCBI using BLAST (
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