ACE2-293T cells transformed with pcDNA 3.1-A29L-His tag, pcDNA 3.1-B6R-His tag, pcDNA 3.1-A35R-His tag, and pcDNA 3.1-M1R-His tag were propagated and harvested, respectively. Cell from cultures were resuspended in Laemelli SDS sample buffer (Thermo Fisher Scientific, Waltham, MA, USA) and heated at 95°C for 10 min. These samples were separated on 12% SDS-PAGE, transferred to PVDF membrane (Sangon Biotech, Shanghai, China) and blocked with 5% skimmed milk-Tris buffered saline amended with 0.1% Tween 20 (TBS-T, Sangon Biotech, Shanghai, China) overnight at 4°C. Then wash three times, membrane was incubated for 1 h with anti-A29L, anti-B6R, anti-A35R and anti-M1R antibodies (Vazyme, Nanjing, China) at RT with continuous slow shaking, respectively. Membrane was incubated with 1:3000 anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at RT. Signals were detected using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA).
Anti rabbit igg conjugated to horseradish peroxidase
Manufactured by Merck Group
Sourced in United States
Anti-rabbit IgG conjugated to horseradish peroxidase is a lab equipment product. It consists of rabbit immunoglobulin G (IgG) molecules covalently linked to the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of rabbit IgG in various experimental and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Ecl reagent, by Promega (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Pvdf membrane, by Sangon (1 mentions)
Labimage 1d l340, by Intas (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (1 mentions)
Phalloidin ifluor 594 reagent, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
6 protocols using anti rabbit igg conjugated to horseradish peroxidase
1
Expression and Detection of VACV Proteins
2
Investigating Nipah Virus Protein Expression
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Polyclonal rabbit antisera against the unique C-terminal domains of the NiV P, W and V proteins and to the NiV C protein were produced by GenScript (Piscataway, NJ). Overall, 1.2 × 106 Vero cells per well were seeded in a six-well plate and infected with rNiVM-wt, rNiVM-Wko or rNiVM-Vko at an MOI of 0.01. The cells were harvested at 40 h.p.i. in 1 ml of 2 × Laemmli sample buffer (Bio-Rad, Hercules, CA) and heated to 95 °C for 20 min. Samples were then run on a denaturing 4–12% SDS–PAGE gel (Bio-Rad). Proteins were transferred from the gel on polyvinilidene fluoride (PVDF) membranes and blocked in TTBS (100 mM Tris-HCl pH 7.5, 0.9% NaCl, 0.1% Tween 20) with 5% skim milk. PVDF membranes were incubated with polyclonal rabbit antisera against P, V, W and C described above, diluted in TTBS with 5% milk (P: 1:10,000; V: 1:5,000; W: 1:5,000; C: 1:500) for 1 h at room temperature and washed three times in TTBS. The membranes were then incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich; 1:20,000 dilution) for 1 h at room temperature, washed three times in TTBS, incubated with ECL reagent (Promega) for 5 min and imaged with a VersaDoc (Bio-Rad).
3
Western Blot Analysis of Recombinant Proteins
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NEB express cells transformed with pp5X+ and pc5X+ were propagated and harvested as described earlier. Equal cell mass from cultures harbouring control and chimeric plasmids were resuspended in SDS loading dye (Laemelli loading buffer) and heated at 95 °C for 7 minutes and cooled on ice for 5 minutes. These samples were separated on 12% SDS-PAGE, transferred to PVDF membrane and blocked with 5% skimmed milk-Tris buffered saline amended with 0.1% Tween-20 (TBS-T) overnight at 4 °C. After blocking the membrane was washed five times with TBS-T and then incubated for 1 hr with anti-MP antibodies (lab raised) at room temperature (RT) with continuous slow shaking. Membrane was again washed several times (five times) with TBS-T and further incubated with 1:5000 anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich, USA) for 30 minutes at RT. Signals were detected using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific, USA).
4
Protein Expression Analysis by SDS-PAGE and Western Blot
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SDS-PAGE and Western blotting of total cell lysates were performed with 4 × 105 cells per lane as described [49 (link),50 (link),51 (link)]. The secondary antibodies used were anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich, Darmstadt, Germany) at a 1:10,000 dilution, followed by chemiluminescence detection. Images were recorded using an Intas ECL Chemostar documentation system. Band intensity and protein molecular mass were determined using LabImage 1D L-340 software (Intas Science Imaging Instruments GmbH, Göttingen, Germany).
5
Immunofluorescence and Western Blot Analysis of Emid1-His
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Cultured cells transfected with Emid1-His were plated on eight-well chamber slides for 24–48 h. Cells were fixed with 4% paraformaldehyde and permeabilized for 10 min at room temperature with 0.1% Triton-X in phosphate buffer saline (PBS). Cells were stained with Anti-His-tag mAb was conjugated with Alexa Fluor 488 (clone OGHis, 1/1000, MBL) for 30 min. Phalloidin-iFluor 594 reagent (Ab176757, 1/1000, Abcam) was used for filamentous actin (F-actin) staining. The slides were mounted in mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Southern biotech) and observed with fluorescence microscopy (Olympus).
Western blotting was performed using cellular protein extracted with cell lysis reagent, deposited protein on the dish collected with a cell scraper, and secreted protein in serum-free medium concentrated with an iCON Concentrator 20K Pierce. Proteins (10 μg) were electrophoresed with standard sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in non-reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). After blocking with 5% skimmed milk for 1 h, the membrane was incubated with polyclonal rabbit antibody EMU1-179, and then incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich). The signals were visualized with enhanced chemiluminescence (ECL Advance Cytiva).
Western blotting was performed using cellular protein extracted with cell lysis reagent, deposited protein on the dish collected with a cell scraper, and secreted protein in serum-free medium concentrated with an iCON Concentrator 20K Pierce. Proteins (10 μg) were electrophoresed with standard sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in non-reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). After blocking with 5% skimmed milk for 1 h, the membrane was incubated with polyclonal rabbit antibody EMU1-179, and then incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich). The signals were visualized with enhanced chemiluminescence (ECL Advance Cytiva).
6
Antibody Detection of Nipah Virus Proteins
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