Left atrial tissue was homogenized in lysis buffer containing radioimmunoprecipitation assay (RIPA) buffer and inhibitors of proteases. Protein concentrations were determined by the bicinchoninic acid (BCA) assay. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), with 8% gels used for endothelial nitric oxide synthase (eNOS) and Collagen I and 10% gels used for TGF-β1, Smad7, and α-SMA. The fractionated proteins were electrophoretically transferred to nitrocellulose membranes (Amersham, Piscataway, NJ, USA). The membranes were incubated with the appropriate primary antibodies for each target protein at 4°C overnight. The primary antibodies for TGF-β1 (1: 500), eNOS (1: 500), Collagen I (1: 500), and α-SMA (1: 200) were from Abcam (Cambridge, MA, USA). The Smad7 (1: 1000) primary antibody was from Aviva Systems Biology (San Diego, CA, USA). The secondary antibodies for Smad7 and eNOS were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Abcam), diluted 1: 5000. The secondary antibodies for TGF-β1, Collagen I, and α-SMA were HRP-conjugated goat anti-mouse IgG (Abcam), diluted 1:5000. Stained bands were visualized after incubation with HRP-conjugated secondary antibody using enhanced chemiluminescence (ECL) detection reagents (Amersham). GAPDH (1: 2500; Solarbio, Beijing, China) was used as a loading control.
Hrp conjugated goat anti mouse igg
Manufactured by Abcam
Sourced in United Kingdom, United States, China
HRP-conjugated goat anti-mouse IgG is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse immunoglobulin G (IgG) in immunoassays and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit igg, by Abcam (7 mentions)
Spectramax 190, by Molecular Devices (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti aβ1 42 antibody, by BioLegend (2 mentions)
Dmi4000b, by Leica (2 mentions)
Supersignal elisa femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Prism 8, by GraphPad (2 mentions)
Stop solution, by Solarbio (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Goat anti rat igg, by Abcam (2 mentions)
Rat anti ha, by Merck Group (2 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Goat anti rabbit igg, by Abcam (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Cell lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
β actin, by Merck Group (2 mentions)
54 protocols using hrp conjugated goat anti mouse igg
1
Western Blot Analysis of Left Atrial Proteins
2
ELISA for SADS-CoV Antibody Detection
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The ELISA plates were coated with 50 μL of the culture supernatants of SADS-CoV-infected Huh7 cells (1:1) diluted in carbonated coating buffer (pH 9.6) at 4 °C overnight. The plates were washed 4 times with PBST (phosphate-buffered saline (PBS) containing 0.05% Tween 20) and blocked with 2% trehalose in PBST at 4 °C for 10 h. For positive hybridoma screen, 50 μL of the supernatants of hybridoma was added to each well. For the titer of the purified anti-N MAb and PAb detection, the antibodies were serially diluted (1:100–1:102,400) in PBST and added into ELISA plates (50 μL/well). The plates were incubated for 30 min at 37 °C. After washing, the HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG (Abcam, Cambridge, MA, USA) diluted at 1:20,000 in PBST was added (50 μL/well) and incubated at 37 °C for 30 min. After washing, 50 μL/well of freshly prepared TMB substrate solution was added and reacted for 10 min at 37 °C in the dark. The reaction was stopped by adding 2 M H2SO4 (50 μL/well). The absorbance was measured at 450 nm by a multifunctional plate reader (BioTek Synergy HTX, Winooski, VT, USA). The supernatant of SP2/0 cells, unrelated MAb, and unimmunized rabbit serum was used as negative control (NC). Sample is considered to be positive if OD450 of sample divided by OD450 of negative control > 2.
3
Investigating Autophagy Regulation Mechanism
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Rotenone, PQQ, trypsin, monoclonal mouse anti-β-actin antibody were from Sigma (St. Louis, MO, USA). Monoclonal anti-LC3 mAb-HRP-DirecT was purchased from MBL (Woburn, MA, USA). Rabbit anti-Atg5 antibody, HRP-conjugated donkey anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were from Abcam (Cambridge, MA, USA). TRIzol reagent, MitoTracker Green (MTG) and LysoTracker Red (LTR) were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA). ELISA kits for IL-1β, IL-6 and TNF-α, cell lysis buffer, protease inhibitor cocktail, BCA-based protein quantification kit, BeyoECL Plus and LDH Cytotoxicity Assay Kit were purchased from Beyotime (Shanghai, China). The Omniscript reverse transcription (RT) kit was purchased from Qiagen (Valencia, CA, USA). 5-ethynyl-2′-deoxyuridine (EdU) Labeling Kit was from Ribobio (Guangzhou, China). Fast EvaGreen qPCR Master Mix was purchased from Biotium (Hayward, CA, USA). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were from Gibco (Grand Island, NY, USA). 3-methyladenine (3-MA) was from MCE (Monmouth Junction, NJ, USA).
4
HA1-specific IgG and IgA ELISA Protocol
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HA1-specific IgG and IgA antibodies were detected by enzyme-linked immunosorbent assay (ELISA) following the protocol described by Joan et al. [28 (link)] with some modifications. Briefly, 96-well microtiter plate (Costar Corporation, USA) was coated with 100 μl of HA1/L/AcmA recombinant protein (1 μg/well) at 4°C for overnight. The wells were blocked with 3% (w/v) BSA for 1 h. Sera or mucosal samples were added and incubated for 1 h. Bound antibody was detected using HRP-conjugated goat anti-mouse IgG (Abcam, UK) at 1:10,000 dilution or HRP-conjugated rat anti-mouse IgA (Abcam, UK) at 1:1,000 dilution. The plates were washed 3 times with PBS between steps. TMB peroxidase substrate system (KPL, USA) was added for detection. After incubation for 20 min, TMB stop solution (KPL, USA) was added and absorbance value at 450 nm was measured using a microplate reader (Tecan, Switzerland). The assay was performed with 3 technical replicates.
5
Recombinant Protein Expression Optimization
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StmΔsadA and StmΔygeAΔmurI strains with the recombinant plasmid were grown aerobically in 5 mL of LB liquid medium (200 μg/mL ampicillin) and LB liquid medium (200 μg/mL ampicillin and 5 mM D-Glu) overnight at 37 °C, respectively. The next day, the cells were inoculated and induced with the addition of 1 mM IPTG when the OD600 value reached about 0.6, and then incubated for 15 h at 16 °C. 1 OD600 bacterial cells were harvested, washed twice with PBS, and resuspended in 200 μL of SDS sample buffer. A 30 μL sample was subjected to 4~12% SDS-PAGE (GenScript, Nanjing, China). The transferred PVDF membrane was blocked in PBST containing 5% skim milk at 4 °C overnight. Subsequently, the membrane was incubated with anti-Flag tag rabbit polyclonal antibody (diluted to 1:3000 with 5% skim milk in PBST; Easybio, Beijing, China), or A1H10 or A3C10 (1:2000 dilution) for 1 h at 37 °C. After three washes with PBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution; Easybio, Beijing, China) or HRP-conjugated goat anti-mouse IgG (1:5000 dilution; Abcam, Cambridge, UK) for 1 h at 37 °C. An ECL-enhanced Western Blot Analysis Kit (Easybio, Beijing, China) was used to detect the binding reactions.
6
Evaluating Antibody Recognition of S. pneumoniae
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Antibody recognition of S. pneumoniae was assessed using previously described whole cell ELISAs and flow cytometry assays (32 (link)). Briefly, for whole cell ELISAs S. pneumoniae were grown to an OD600 of approximately 0.4–0.8, washed and resuspended to an OD600 of 0.4 in PBS, 50 μl/well were added to microtiter plates and incubated overnight at RT before fixation in 4% formaldehyde for 10 min. Plates were washed and incubated with a 1:100 dilution of murine antiserum for 1 h at 37°C and using HRP-conjugated goat anti-mouse IgG (abCam, ab6789) for detection. For flow cytometry antibody binding assays live S. pneumoniae (1 x 106 CFU) were incubated for 30 min at 37°C with 10% mouse serum. Fluorescently labelled anti-mouse IgG (BioLegend, 405308) and IgM (BioLegend, 406505) were used to detect antibody binding to the bacterial surface using a BD FACSVerse instrument.
7
ELISA Protocol for Antibody Titer Determination
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96-well EIA/RIA plates (Corning) were coated overnight at 4 °C with 20 μg of G protein per plate in coating buffer (Solarbio). All wells were blocked with 100 µl of blocking buffer (5% skim milk in PBS) for 1.5 h at 37 °C. After standard washes and blocks, serum (2x serially diluted, starting at 100x dilution) in 5% skim milk in PBS was incubated at 37 °C for 2 h. Plates were washed four times in PBS with 0.1% Tween 20 (PBST) before addition of HRP-conjugated goat anti-mouse IgG (Abcam, Cat No. ab6789, diluted 1:20,000), IgA (Abcam, Cat No. ab97235, diluted 1:10,000) or HRP-conjugated goat anti- hamster IgG (Abcam, Cat No. ab6892, diluted 1:15,000) diluted in 5% milk in PBS for 1.5 h at 37 °C. Plates were washed as before prior to being developed with 100 µl/well of TMB chromogen solution (Beyotime) for 15 min. Substrate reactions were stopped by the addition of 50 µl/well of stop solution for TMB Substrate (Beyotime) before reading plate absorbance at 450 nm (OD450). The cutoff value was defined as 2.1-fold of OD450 values from the sample of nonvaccinated mice. The reciprocal of the maximum sample dilution with OD450 values equal to or greater than the cutoff value was used to calculate the endpoint binding antibody titers.
8
Protein Expression Analysis in Brain Cortex
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Brains were immediately dissected after euthanasia, and the cortex was separated on ice and stored at −80°C. The total protein in the cortex was obtained with a whole protein extraction kit (cat. no. KGP250; Nanjing KeyGen Biotech Co., Ltd.) and then quantified using a bicinchoninic acid assay kit (Boster Biological Technology). A total of 40 µg of each protein sample was loaded per lane and then separated via 8% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). After blocking in TBS-0.1% Tween-20 and 5% nonfat dry milk at room temperature for 1 h, the membranes were incubated with rabbit polyclonal anti-ZO-1 antibody (1:1,000; cat. no. 61-3700; Zymed; Thermo Fisher Scientific, Inc.), rabbit anti-HSP70 antibody (1:1,000; cat. no. ab181606; Abcam) and mouse anti-GAPDH antibody (1:2,000; cat. no. ab8245; Abcam) at 4°C overnight. After rinsing with TBS-0.1% Tween 20, membranes were then incubated HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ab6721; Abcam) and HRP-conjugated goat anti-mouse IgG (1:5,000; cat. no. ab6789; Abcam) at room temperature for 2 h. Finally, the membranes were washed four times and developed using an Immobilon Western Chemiluminescent HRP Substrate kit (EMD Millipore). Gray value analysis was performed using QuantityOne software version 4.6.8 (Bio-Rad Laboratories, Inc.).
9
Quantification of L1 Endonuclease Protein
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8 × 106 cells were lysed in M-PER buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (Roche). 50 μg of whole cell extract were used for immunoblotting as previously described [26 (link)]. mAb chA1-L1 (0.7 μg/ml) was used as the primary antibody. For the peptide competition assay, membranes were probed with an equimolar mixture of chA1-L1 and peptide 39, pre-incubated for 45 minutes at room temperature. The sensitivity of the antibody was assessed by immunoblot analysis of increasing amounts (1 to 75 ng) of the purified L1 endonuclease domain (L1-EN) [34 (link)] added to 50 μg of WI-38 whole cell extracts. HRP-conjugated goat anti-mouse IgG (Abcam) was used as the secondary antibody. Membranes were probed with an anti-α-tubulin antibody (Sigma-Aldrich) to verify equal loading.
10
Western Blot Analysis of Adenovirus-Infected Cells
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OFTU cells were infected with recombinant adenovirus (MOI = 100) cells were harvested after 48 h, PMSF and RIPA lysis buffer (Beyotime, Shanghai, China) were mixed (1:100) and added separately to prevent protein degradation. Proteins were electrophoresed on a 10% separating gel; proteins were transferred to nitrocellulose membranes; mouse polyclonal antibodies (22 (link)) in the ratio of 1:500 or GAPDH mouse monoclonal antibody (1:5000) (Proteintech, Chicago, USA) was used as the primary antibody and incubate overnight at 4°C. HRP-conjugated goat anti-mouse IgG (1:5000) (Abcam, Cambridgeshire, UK) was used as the secondary antibody and incubated at 37°C for 1 h. Record results using a gel imaging system (Tanon, Shanghai, China).
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