Confirmation of protein identities was performed by Western blotting. Protein extracts were prepared in lysis buffer and each cell lysate sample (1 μg/μL, 10 μL) was electrophoresed through a precast gel (NuPAGE®Novex® 4–12% Bis-Tris Gel, 1.5 mm, 10 wells, Invitrogen™, Carlsbad, CA). Proteins were transferred from the gel to a polyvinyldifluoride (PVDF) membrane (Millipore, Bedford, CA) by means of the semidry technique using the Criterion Blotter (Bio-Rad) at 100 V for 60 min and blocked with 5% milk in PBS (adjusted to pH 7.4) containing 0.05% Tween-20. The membranes were then separately incubated overnight with primary rabbit antibodies (1 μg/μL). The commercially available primary antibodies used in this study included the following: monoclonal mouse anti-HSP60 (Stressgen, USA) and polyclonal rabbit anti-RanBP2 (Abcam, USA). After washing, the membrane was incubated with HRP-conjugated goat anti-mouse IgG antibodies (purchased from Jackson ImmunoResearch, USA) for 1 hour (1 : 10000). Proteins were detected with an enhanced chemiluminescent (ECL) system, and quantitative analysis of Western blotting was carried out using the ImageQuant-TL-7.0 software, version 2010 (Amersham Biosciences).
Hrp conjugated goat anti mouse igg antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States, Panama, United Kingdom
The HRP-conjugated goat anti-mouse IgG antibody is a secondary antibody used for the detection of mouse immunoglobulin G (IgG) in various immunoassay applications. It is conjugated with horseradish peroxidase (HRP), which serves as a reporter molecule, enabling the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Chemiluminescence hrp substrate, by Merck Group (2 mentions)
Las 3000 image system, by Fujifilm (2 mentions)
Bm blue pod substrate soluble, by Roche (2 mentions)
Uv 2401pc, by Shimadzu (2 mentions)
Criterion blotter, by Bio-Rad (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Monoclonal mouse anti hsp60, by Stressgen (1 mentions)
Polyclonal rabbit anti ranbp2, by Abcam (1 mentions)
Imagequant tl 7.0 software version 2010, by Cytiva (1 mentions)
Immobilon pvdf, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Jackson ImmunoResearch (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti myc, by Merck Group (1 mentions)
Anti egfp, by Roche (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti gadd45α, by Santa Cruz Biotechnology (1 mentions)
23 protocols using hrp conjugated goat anti mouse igg antibody
1
Western Blotting Protocol for Protein Identification
2
Western Blot Analysis of B. pertussis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ten micrograms of crude bacterial protein extract of B. pertussis grown in the presence (Bp+Fe) or absence (Bp-Fe) of iron were resolved by 7.5% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (Immobilon PVDF Millipore) sheets [28] and incubated with mouse anti-FHA (1:500) or mouse anti-CyaA (1:500) antibodies. HRP-conjugated goat antimouse IgG antibodies (Jackson ImmunoResearch, Baltimore Pike, 1:2500) were used for immunochemical detection. An ECL Western Blotting Detection Reagent (Pierce Biotechnology, Rockford, IL, USA) was used to detect specific signals.
3
SARS-CoV-2 Neutralization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vero cells (ATC-CCL81) cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, catalog no. D6546) with 10% FCS, 2 mM l -glutamine (Sigma-Aldrich, G7513), and 1% penicillin/streptomycin (Invitrogen, 15140148) were seeded the day prior to infection at 2 × 104 cells per well in a 96-well flat-bottom plate. Serial dilutions of proteins of interest were incubated with 100 TCID50 of SARS-CoV-2 (strain England/02/2020) for 1 h at 37°C, 5% CO2. After careful removal of culturing medium from Vero monolayer, 100 μl of protein-virus mixtures was added to the cells and incubated 1 h at 37°C, 5% CO2. Subsequently, 100 μl of culturing medium with 4% FBS was added to occupied wells, and plates were incubated at 37°C, 5% CO2 for 48 h. After removal of culturing medium, occupied wells were fixed with 4% paraformaldehyde (PFA) in PBS for 1 h at room temperature for viral inactivation, followed by incubation with 0.1% Triton X-100 for 15 min at room temperature for cell permeabilization. Plates were washed with 0.05% vol/vol PBS-Tween and sequentially incubated with mouse anti-SARS-CoV-2 N protein antibody (The Native Antigen Company, MAB12183-100) at 1:500 dilution and HRP-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch, 115-035-146) at 1:5,000 dilution in 3% wt/vol milk in 0.05% PBS-Tween. Plates were acquired on a BMG FLUOstar Omega at a wavelength of 450 nm.
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% IGEPAL CA-630, 1 mM phenymethylsulfonyl fluoride (PMSF), 20 mg/ml pepstatin A, 20 mg/ml leupeptin, 20 mg/ml aprotinin, 50 mM NaF and 1 mM Na3VO4] and the protein lysates were harvested by centrifugation at 14,000 rpm to remove the debris. The lysate was resolved by 10% or 12% SDS-PAGE and proteins separated by SDS-PAGE were transferred onto the PVDF membrane for antibody conjugation. The antibodies used include: anti-Flag M2 (Sigma-Aldrich, St. Louis, MO), anti-actin (Millipore), anti-EGFP (Roche, Penzberg, Germany), anti-GADD45α (Santa Cruz Biotechnology, Santa Cruz, CA), anti-SUMO-1 (Active Motif), anti-SUMO-2 (Epitomics, Burlingame, CA), anti-Hes-1 (GeneTex), anti-pSer263 Hes-1
[3 (link)], anti-PIAS1 (Epitomics), anti-pSer90PIAS1 (LTK BioLaboratories, Taoyuan, Taiwan), anti-α-His (Millipore) and anti-Myc (Millipore) antibodies. The secondary antibodies used were HRP-conjugated goat-anti-mouse IgG antibody and HRP-conjugated goat-anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Membrane was developed by reacting with chemiluminescence HRP substrate (Millipore) and exposed to the LAS-3000 image system (Fujifilm, Tokyo, Japan) for visualization of protein bands.
[3 (link)], anti-PIAS1 (Epitomics), anti-pSer90PIAS1 (LTK BioLaboratories, Taoyuan, Taiwan), anti-α-His (Millipore) and anti-Myc (Millipore) antibodies. The secondary antibodies used were HRP-conjugated goat-anti-mouse IgG antibody and HRP-conjugated goat-anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Membrane was developed by reacting with chemiluminescence HRP substrate (Millipore) and exposed to the LAS-3000 image system (Fujifilm, Tokyo, Japan) for visualization of protein bands.
5
ELISA Assay for Mouse Antibody Titers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified MCF was coated on an ELISA plate at 20 μg/ml. Then, the plate was washed with 1X phosphate-buffered saline + 0.05% Tween-20 (PBST) three times and blocked in 1% bovine serum albumin in phosphate-buffered saline (BSA-PBS). For monitoring mouse antibody titers, mouse serum was diluted 1:1,000 in 1% BSA-PBS, then diluted two-fold up to 1:128,000, and allowed to incubate on the ELISA plate for 1 h. For testing hybridoma cultures, neat supernatant was added directly to the blocked ELISA plate and incubated for 1 h. After primary antibody incubation, plates were washed three times with PBST and then incubated with horseradish peroxidase (HRP)-conjugated goat-anti mouse IgG antibody (1:5,000 dilution, Jackson ImmunoResearch, West Grove, PA, United States) for 1 h. Plates were washed four times with PBST and TMB substrate was added. After color development, the reaction was stopped with 0.16 M sulfuric acid, and absorbance was read at 450 nm.
6
Western Blot Analysis of Plant-Produced rhuEPO
7
Quantification of Amyloid-Beta Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentration of lysate was quantified by Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA). The clarified lysate (16 μg) was mixed with 5× protein loading buffer (GeneMark) before boiling at 95 °C for 10 min and were subjected to 13% Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring onto the polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Before blocking with 5% skim milk, membrane was submerged in boiling phosphate-buffered saline (PBS) for 6 min. Immunoblot was conducted using the mouse anti-human amyloid-beta 6E10 antibody (1:3000, BioLegend, San Diego, CA). The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:6500, Jackson ImmunoResearch, West Grove, PA). Membrane was developed by reacting with chemiluminescence HRP substrate (Millipore) and exposed to the LAS-3000 image system (Fujifilm, Tokyo, Japan) for visualization of protein bands. The protein bands were quantified using the NIH Image J Software.
8
PDGF-B Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 23
Human recombinant PDGF-B was immobilized on a 96-well NUNC Maxisorb plate at 2.5 μg/ml (mouse) or 1.0 μg/ml (rabbit), 100 μl/well, in PBS, followed by: blocking of the plate with 2% CroteinC in PBS, 200 μl/well; application of serial dilutions of antisera, in duplicates, in 0.5% CroteinC in PBS, 100 μl/well; detection with HRP-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch) diluted 1:16,000 in 0.5% CroteinC in PBS for mouse sera or with biotinylated goat anti-human kappa antibody (Southern Biotech) diluted 1:5,000 and HRP-conjugated streptavidin diluted 1:8,000 in 0.5% CroteinC in PBS for rabbit sera, 100 μl/well. For all steps, plates were incubated for 1 hour at 37° C. Between all steps, plates were washed 3 times with 0.05% Tween 20 in PBS. Signal was developed by addition of BM Blue POD Substrate soluble (Roche Diagnostics GmbH, Mannheim, Germany), 100 μl/well; and stopped by addition of 1 M HCl, 100 μl/well. Absorbance was read out at 450 nm, against 690 nm as reference. Titer was defined as dilution of antisera resulting in half-maximal signal.
9
Antibody-Based Biochemical Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glyceryl triacetate, buffer reagents, β-glycerophosphate, erythro-9-(2-hydroxy-3-nonyl) adenine, α, β-methyleneadenosine 5’-diphosphate, and 2-mercaptoethanol were purchased from Sigma (Sigma, St. Louis, MO, USA). A mouse anti-human CD73 antibody and a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody were obtained from AbD Serotec (AbD Serotec, Raleigh, NC, USA). A HRP-conjugated goat anti-mouse IgG antibody was purchased from Jackson ImmunoResearch (Jackson ImmunoResearch, Westgrove, PA, USA). A mouse anti-adenosine receptor A2A antibody was obtained from Upstate (Upstate, Billerica, MA, USA). A goat anti-adenosine kinase antibody, mouse anti-α tubulin antibody, HRP-conjugated donkey anti-goat IgG antibody, and HRP-conjugated goat anti-mouse IgM antibody were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Absolute ethanol was from Pharmco (Pharmco, Brookfield, CT, USA).
10
Sandwich ELISA for Quantifying HtrA1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Standard sandwich ELISAs were performed using HtrA1-specific NEP-2717 polyclonal antibody for capture and B18 monoclonal anti-HtrA1 antibody for detection, followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch). HtrA1 antibody generation and characterization along with full details of the ELISA are described in the SI Appendix .
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!