The viral proteins in each fraction were separated by 5–20% e-PAGEL (Atto, Tokyo) and then stained with Coomassie brilliant blue (CBB). The proteins in the gel were electrophoretically transferred onto a nitrocellulose membrane. The membrane was then blocked with PBS-T containing 5% skim milk and incubated with a rabbit hyperimmune antiserum against HuSaV GII.3 C12 VP1–VLPs (1:1000) in PBS-T containing 1% skim milk [7 (link)]. Detection of the rabbit IgG antibody was achieved using alkaline phosphatase-conjugated goat anti-rabbit antibody (1: 1000) (Chemicon International, Billerica, MA, USA). Nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine were used for the detection of antibody binding (Bio-Rad Laboratories).
Nitroblue tetrazolium chloride
Manufactured by Bio-Rad
Sourced in United States
Nitroblue tetrazolium chloride is a chemical compound used in various biochemical and molecular biology applications. It serves as an indicator for the detection and visualization of certain enzymatic activities, particularly those involving redox reactions. The compound undergoes a color change when reduced, making it a useful tool for experimental procedures in the laboratory setting.
Automatically generated - may contain errors
5 protocols using nitroblue tetrazolium chloride
1
Immunodetection of Viral Proteins
2
Western Blot Detection of Viral Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The viral proteins were detected by Western blot analysis (WB). The virus-infected PLC/PRF/5 cells or pET32a-ORF4-transformed BL21 (DE3) were separated by centrifugation at 10,000× g for 5 min and suspended with PBS. The viral proteins were separated by 5–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% skim milk in 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, and incubated with cynomolgus monkey anti-HEV-1 serum (1:500 dilution) with PBS-T containing 1% skim milk [25 (link)]. Detection of the monkey IgG antibody was achieved using alkaline phosphatase-conjugated rabbit anti-monkey IgG (whole molecular) (1:1000 dilution) (Sigma-Aldrich, St. Louis, MO, USA). Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate P-toluidine were used as the coloring agents (Bio-Rad Laboratories, Hercules, CA, USA).
3
Detecting G5 Hepatitis E Virus Capsid Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The capsid protein of G5 HEV was detected by western blot (WB) analysis. The culture supernatant of the G5 HEV–infected PLC/PRF/5 cells was separated by centrifugation at 10,000g for 30 minutes, and the supernatants were further concentrated by ultracentrifugation at 100,000g for 2 hours in a Beckman SW 55 Ti rotor. The pellet was resuspended in PBS. The viral proteins were separated by 5% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% skim milk in 50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl and incubated with rabbit anti‐G5 HEV‐LPs polyclonal antibody (1:1,000 dilution with PBS‐T containing 1% skim milk). Detection of the rabbit IgG antibody was achieved using alkaline phosphatase–conjugated goat anti‐rabbit immunoglobulin (1:1,000 dilution) (Chemicon International, Temecula, CA). Nitroblue tetrazolium chloride and 5‐bromo‐4‐chloro‐3‐indolyl phosphate P‐toluidine were used as the coloring agents (Bio‐Rad Laboratories, Hercules, CA).
Immunofluorescence assay (IFA) with rabbit anti‐G5 HEV‐LPs hyperimmune serum (1:1,000 dilution) was used to detect the G5 HEV capsid protein in the cells as described.17 , 20
Immunofluorescence assay (IFA) with rabbit anti‐G5 HEV‐LPs hyperimmune serum (1:1,000 dilution) was used to detect the G5 HEV capsid protein in the cells as described.
4
Detecting Viral Protein Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The viral proteins in each fraction were separated by SDS-PAGE using 5%–20% e-Pagel (ATTO, Tokyo) and then stained with Coomassie blue. For Western blotting, the separated proteins were electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, and then incubated with 1:500 diluted monkey serum. Detection of the monkey IgG antibody was achieved using phosphatase-labeled goat anti-monkey IgG (H + L) (1:1000 dilution) (abcam, Tokyo). Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate P-toluidine were used as coloring agents (Bio-Rad Laboratories, Hercules, CA)16 (link).
5
Western Blot Detection of MCPyV VP1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins in the cell lysates and culture media were separated by 12.5% SDS—PAGE and were electrophoretically transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% skim milk in 50 mM Tris—HCl (pH 7.4), 150 mM NaCl, and reacted with a rabbit anti-MCPyV VP1 polyclonal antibody. Detection of rabbit IgG antibody was achieved by using alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (Chemicon International). Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate P-toluidine were used as coloring agents (Bio-Rad Laboratories). The MCPyV VP1 antigen to generate a rabbit anti-MCPyV VP1 polyclonal antibody was prepared as follows. A glutathione S transferase-MCPyV VP1 fusion protein was bacterially produced from pGEX5X/MCPyVVP1, where MCPyV VP1 gene was inserted into the BamHI-XhoI site of pGEX5X-2 (GE Healthcare), and purified by glutathione Sepharose chromatography.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!