The EV-A71-infected and gemcitabine-treated RD cells were incubated for 8 h before total cellular proteins were extracted using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, #78501) containing protease inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific, #78440) and EDTA. Proteins were separated in 10% polyacrylamide gel and transferred to nitrocellulose membranes using the Bio-Rad semidry transfer system. Mouse anti-EV-A71 VP2 protein (Millipore, #MAB979) was used to detect EV-A71 VP0 and VP2 proteins, mouse anti-EV-A71 3D protein (GeneTex, #GTX630193) was used to detect EV-A71 3D and 3CD proteins and mouse anti-actin (Millipore, #MAB1501) was used as loading control. Goat anti-mouse IgG conjugated to HRP (Thermo Fisher Scientific, #31430) was used as the secondary antibody.
Goat anti mouse igg conjugated to hrp
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-mouse IgG conjugated to HRP is a secondary antibody used in various immunoassay techniques. It is designed to detect and bind to mouse primary antibodies, with the horseradish peroxidase (HRP) label enabling colorimetric or chemiluminescent detection.
Automatically generated - may contain errors
Lab products found in correlation
Ab6308, by Abcam (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Flag tag (flag), by Beyotime (1 mentions)
Immulon 4hbx plate, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
7 protocols using goat anti mouse igg conjugated to hrp
1
EV-A71 Protein Detection in Gemcitabine-Treated RD Cells
2
Western Blot Analysis of Fusion Proteins
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Purified baculoviruses and infected cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Primary antibodies (1:4000 dilution), including against 6×His (Beyotime, Shanghai, China), V5 (Thermo Fisher, Waltham, MA, USA), and Flag (Beyotime, Shanghai, China) tags, were used to detect fusion proteins by western blot analysis. The secondary antibodies were goat anti-pig and goat anti-mouse IgG conjugated to HRP (1:3000 dilution, Thermo Fisher, Waltham, MA, USA). Protein bands were visualized using the ECL chemiluminescence system and Hyper-Max films, as recommended by the manufacturer.
3
Collagen Type 1 Quantification Assay
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Human Met5a cells were plated into a 96‐well plate at 70,000 cells per well. The plates were incubated at 37°C for 3 h. The cells were then treated with pooled sera according to their treatment group designation which can be described as follows: No treatment, MCAA+, anti‐PLG, and MCAA+ serum cleared of anti‐PLG. The cells were then incubated for 82 h at 37°C and rinsed once with PBS. All blocking and incubation steps were performed under gentle agitation at room temperature for 1 h with washing steps occurring immediately following the incubation steps. The wells were then blocked with 200 μL 5% nonfat dry milk in PBS. The wells were incubated with 2 μg of mouse IgG targeted to collagen type 1 (ab6308; Abcam, Cambridge, MA) in 100 μL of 3% BSA. The plates were then washed 3× with 200 μL of 0.05% Tween in PBS for 4 min each wash. The plates were blocked again with 5% milk in PBS. The plates were then incubated with goat anti‐mouse IgG conjugated to HRP (Life Technologies). The plates were then washed 3× with 200 μL of 0.05% Tween in PBS. The plates were developed with 100 μL of Pierce One Step TMB ELISA reagent. The reactions were stopped with 50 μL of 1 mol/L HCl. The plates were then read for absorbance at 450 nm on the BioTek plate reader.
4
Quantifying Collagen Type 1 Immunoassay
5
ELISA for Lyme Disease Antibody Detection
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B. burgdorferi whole-cell lysate was prepared as previously described (53 (link)). Total protein in the sonicate was quantified on the basis of the absorbance at 280 nm using a NanoDrop1000 spectrophotometer (Thermo Scientific), and aliquots were stored at −20°C. The protein was diluted to 5 µg/mL, and 50 µL per well was placed in Immulon 4HBX plates (Thermo Scientific) and coated overnight at 4°C. After blocking with 3% BSA, serially diluted serum was added in PBS with 2% BSA and incubated for 2 h at RT. Goat anti-mouse IgG conjugated to HRP (cat #A16084, Invitrogen) or goat anti-mouse IgM conjugated to HRP (cat #62–6820, Invitrogen) was diluted to 1:5,000 or 1:2,000, respectively, in 1% BSA and incubated for 1 h at RT. After incubation, antigen-specific antibodies were detected using TMB substrate as described by the manufacturer (Thermo Scientific), and the reaction was stopped with 2-M H2SO4. Antibody endpoint titers for IgG were determined as the highest serum dilution corresponding to a cutoff of 1.0 Abs450. Antibody endpoint titers for IgM were determined as the highest serum dilution corresponding to a cutoff of 0.2 Abs450.
6
Monoclonal Antibody Specificity Validation
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To confirm specificity of each monoclonal antibody, an ELISA was performed. Synthetic peptides IhfA3, IhfA5, IhfB2 and mIhfB4 in addition to purified IHFNTHI α- and β-subunit proteins were suspended in carbonate-bicarbonate buffer (pH 9.6), 1.0 μg added to each well of MaxiSorp C-bottom microtiter plates (Nunc) and adsorbed for 18 h at 4 °C. Wells were then blocked with 2% bovine serum albumin (Sigma) in 10 mM phosphate buffered saline (pH 7.4; PBS), washed with 10 mM PBS and followed by addition of 0.1 μg monoclonal antibody per well. Wells were washed as before and goat anti-mouse IgG conjugated to HRP (Invitrogen; RRID AB_11180340 ) next added. Colour was developed with One-Step Ultra TMB (Pierce) and the reaction stopped with 2 M H2SO4. Assays were repeated a minimum of three times, on separate days. A representative assay is shown in Fig. 1 B.
7
Western Blot Protein Detection
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The infected cell lysates were subjected to gradient sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis (PAGE) gel (HR gradient gel solution, TOOLS, Taipei, Taiwan). After electrophoresis, proteins were transferred to PVDF membranes. The protein signals were detected by using mouse anti-6xHis-tag monoclonal antibody (1:5000 dilution, EnoGene, New York, NY, USA). Then, the goat anti-mouse IgG conjugated to HRP (1:5000 dilution, Invitrogen, Carlsbad, CA, USA) were used as the secondary antibodies for signal detection. The protein bands were detected by using the Clarity™ Western ECL Blotting Substrates (Bio-Rad, Hercules, CA, USA) using Classic Blue Autoradiography film BX (Life Science, Valley Park, MO, USA).
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