To confirm the correct expression of the human Tau4R2N or Tau3RC antigens present in MVA-Tau4R2N or MVA-Tau3RC, DF-1 cells were mock-infected or infected with MVA-Tau4R2N, MVA-Tau3RC, and MVA-Δ-GFP at 5 plaque forming units (PFUs)/cell and at 3, 6 and 24 h postinfection (hpi), cell extracts or supernatants were collected, lysed in Laemmli buffer, fractionated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and analyzed by Western blotting with a mouse monoclonal antibody against the microtubule-binding region of the human tau protein (antibody 7.51; kindly provided by Dr. C. M. Wischik, Aberdeen, UK; diluted 1:100) to evaluate the expression of the human Tau4R2N or Tau3RC proteins. As loading controls, a rabbit anti-β-actin antibody (Cell Signaling, Danvers, MA, USA; diluted 1:1000), and a rabbit anti-VACV E3 antibody (CNB; diluted 1:1000) were used. The membranes were incubated with the antibodies at 4 °C overnight. Anti-mouse horseradish peroxidase (HRP)-conjugated antibody (Sigma-Aldrich, St. Louis, MO, USA; diluted 1:2000), or anti-rabbit HRP-conjugated antibody (Sigma-Aldrich, St. Louis, MO, USA; diluted 1:5000), were used as secondary antibodies. The immunocomplexes were detected using an HRP-luminol enhanced-chemiluminescence (ECL) system (ECL Plus) (GE Healthcare, Chicago, IL, USA).
Anti mouse horseradish peroxidase hrp conjugated antibody
Manufactured by Merck Group
Sourced in United States
Anti-mouse horseradish peroxidase (HRP)-conjugated antibody is a laboratory reagent used for the detection and quantification of mouse proteins in various immunoassays. The antibody is conjugated with the enzyme horseradish peroxidase, which catalyzes a colorimetric or chemiluminescent reaction, providing a means to visualize and measure the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti β actin antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit antibody, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ha tag, by Santa Cruz Biotechnology (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Dmi4000b fluorescence microscope, by Leica (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Pro prep protein lysis buffer, by iNtRON Biotechnology (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Anti syntaxin 4 rabbit polyclonal antibody, by Synaptic Systems (1 mentions)
5 protocols using anti mouse horseradish peroxidase hrp conjugated antibody
1
Validating Tau4R2N and Tau3RC Antigens
2
Cell Culture Conditions and Reagents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prof. Yan-Ming Zhang kindly offered SUVEC, College of Veterinary Medicine, Northwest A&F University (19 (link)), was cultured in 10% fetal bovine serum (FBS; Gibco) at 5% CO2, 37 °C. Staurosporine (STS) was purchased from Sigma-Aldrich (MO, USA). HA-tag and MYC-tag monoclonal antibodies were bought from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-mouse horseradish peroxidase (HRP)-conjugated antibody was obtained from Sigma-Aldrich.
3
Indirect Fluorescent Antibody and Immunoperoxidase Assays for BVDV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An indirect fluorescent antibody (IFA) assay was performed using MDBK cells deposited over glass coverslips. Acetone-fixed cells were incubated for 1 h at 37 °C with a pool of monoclonal antibodies (MAbs) to BVDV (15c5, 12g4, and 20.10.6),23 (link) followed by washing in phosphate-buffered saline (PBS) and incubation with an anti-mouse IgG fluorescein isothiocyanate-conjugated secondary antibody (1:100 in PBS; Sigma–Aldrich). Slides were examined under ultraviolet light in a DMI 4000B fluorescence microscope (Leica, Wetzlar, Germany). Immunoperoxidase (IPX) staining for BVDV antigens was performed using cell monolayers grown in six-well plates and inoculated the respective viruses. Cell monolayers were fixed in 30% cold acetone by 13 min and incubated with the same MAbs for IFA (1 h at 37 °C), washed, and incubated with an anti-mouse horseradish peroxidase (HRP)-conjugated antibody (1:1000 in PBS; Sigma–Aldrich). After washing, an HRP substrate (aminoethylcarbazole – AEC, in acetate buffer 50 mM – pH 5.0 and hydrogen peroxide 0.2%) was added and incubated for approximately 30 min at 37 °C.
4
Acanthamoeba-Specific Antibody Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acanthamoeba-specificity of the antibody was determined by western blotting. Briefly, culture media of Acanthamoeba, and whole cell lysates of HCE cells, Acanthamoeba spp., P. aeruginosa, S. aureus, and F. solani were prepared using the Pro-Prep protein lysis buffer (iNtRON BioTechnology, Seongnam, Korea). Protein concentrations were determined using the Bradford reagent (BIO-RAD, CA, USA). Proteins were resolved by SDS-PAGE and transferred on to a nitrocellulose (NC) membrane. The membrane was blocked with 5% skim milk in TBST (25 mmol/L Tris base, 150 mmol/L NaCl, and 0.1% Tween 20) buffer for 1 h at RT, and incubated overnight at 4 ℃ with the IPNH polyclonal antibody. On the next day, the membrane was washed for 5 minutes with TBST three times and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. Membranes were washed and after exposure to enhanced chemiluminescence (ECL; Thermo Fisher, MA, USA), protein expressions were detected on an x-ray film in the darkroom.
5
Antibody Characterization for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Commercially obtained antibodies were as follows: anti-c-Myc (9E10) mouse monoclonal antibody (Santa Cruz Biotechnology); anti-calcium/calmodulin-dependent protein kinase II α (6G9) mouse monoclonal antibody, anti-GAPDH (6C5) mouse monoclonal antibody, and anti-MAP2 chick polyclonal antibody (Millipore Corp.); anti-GFP (clones 7.1 and 13.1) mouse monoclonal antibody (Roche Applied Science); anti-Syntaxin-4 rabbit polyclonal antibody (Synaptic Systems); IRDye®680RD/800CW-conjugated anti-mouse/rabbit IgG donkey antibody (LI-COR Biotechnology); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Sigma); and Cy2/Cy3/Cy5 or Alexa Fluor® 488/594/647-conjugated anti-mouse/rabbit/chick/sheep IgG donkey antibody (Jackson ImmunoResearch). Anti-Rab17 rabbit polyclonal and anti-Myc sheep antibodies were prepared as described previously (18 (link), 19 (link)).
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!