Antibodies with the following specificities were used for Western blot analysis: TRAF2, β-actin (abcam, Cambridge, UK), Caspase-3, cIAP1, cIAP2 (Cell signaling, Danvers, MA, USA), Caspase-8 (Enzo Life Sciences, Lausen, Switzerland), PARP, Cul3 (BD Pharmingen, Heidelberg, Germany), α-Tubulin (Millipore, Billerica, MA, USA) and RIP3 (Imgenex, San Diego, CA, USA). HRP-conjugated goat anti-mouse IgG1, goat anti-mouse IgG2a, goat anti-mouse IgG2b and donkey anti-rabbit antibodies were obtained from Southern Biotech (Birmingham, AL, USA) and Dianova (Hamburg, Germany). Killer-TRAIL was purchased from Alexis biosciences (Carlsbad, CA, USA). The pan-caspase-inhibitor zVAD-fmk was obtained from Bachem (Heidelberg, Germany). Necrostatin-1 was purchased from Enzo Life Sciences (Lausen, Switzerland). Fc-CD95L and Flag-TWEAK were produced as previously described.33 (link) TRAF2 siRNA (HS-TRAF2_4, sequence as follows: 5′-GGACCAAGACAAGAUUGAATT-3′), alternate TRAF2 siRNA's (HS-TRAF2_5, sequence: 5′-CGAGGGCAUAUAUGAAGAATT-3′ HS TRAF2_6, sequence: 5′-GUUCGGCCUUCCCAGAUAATT-3′ HS TRAF2_7, sequence: 5′-GCUGCGGAGCAGACGUGAATT-3′) and negative control siRNA were provided by Qiagen (Hilden, Germany). siRNA transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
Hrp conjugated goat anti mouse igg1
Manufactured by Southern Biotech
Sourced in United States
HRP-conjugated goat anti-mouse IgG1 is a secondary antibody that binds to the IgG1 subclass of mouse immunoglobulins. It is conjugated with horseradish peroxidase (HRP), which can be used for detection and quantification applications in various immunoassays.
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Lab products found in correlation
Hrp conjugated goat anti mouse igg, by Southern Biotech (2 mentions)
Igg2b, by Southern Biotech (2 mentions)
Igg2a, by Southern Biotech (2 mentions)
Hrp conjugated goat anti mouse igg2a, by Southern Biotech (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Necrostatin 1, by Enzo Life Sciences (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Ciap1, by Cell Signaling Technology (1 mentions)
Ciap2, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg2b, by Southern Biotech (1 mentions)
Caspase 8, by Enzo Life Sciences (1 mentions)
Goat anti mouse igg2a, by Southern Biotech (1 mentions)
Zvad fmk, by Bachem (1 mentions)
Maxisorb plate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Sector imager 2400 system, by Mesoscale (1 mentions)
Multiskan mk3, by Thermo Fisher Scientific (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Southern Biotech (1 mentions)
10 protocols using hrp conjugated goat anti mouse igg1
1
Western Blot Analysis of Cell Death Regulators
2
Serum Antibody and Cytokine Profiling
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Blood was collected via cardiac puncture and serum isolated by centrifugation.
H107-specific antibody detection was performed by ELISA. Maxisorb Plates (Nunc) were coated overnight at 4°C with H107 (0.1 μg/ml) and then blocked for with 2% BSA in PBS for 2hrs at room temperature. Sera were diluted 30X followed by three-fold serial dilutions and incubated 2hr at room temperature 4°C on plates. Total IgG was detected with horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG (Invitrogen) diluted 1:32000. IgG1 was detected with HRP-conjugated goat anti-mouse IgG1 (Southern Biotech) diluted 1:16000. IgG2c was detected with HRP-conjugated rabbit anti-mouse IgG2c (Southern Biotech) diluted 1:5000. The enzyme reaction was developed with 3,3’,5,5’- tetramethylbenzidine, hydrogen peroxide (TMB Plus; Kementec), stopped with 0.2 M H2SO4 solution and plates read at 450 nm with 620 nm background correction using an ELISA reader (Tecan Sunrise).
The Mouse U-plex kit for the cytokines IL-12p70, IL-6, TNFα, MCP-1 was performed according to the manufacturer’s instructions (Meso Scale Discovery) to measure cytokine concentrations in serum. The plates were read on the Sector Imager 2400 system (Meso Scale Discovery) and calculation of cytokine concentrations in unknown samples was determined by 4-parameter logistic non-linear regression analysis of the standard curve.
H107-specific antibody detection was performed by ELISA. Maxisorb Plates (Nunc) were coated overnight at 4°C with H107 (0.1 μg/ml) and then blocked for with 2% BSA in PBS for 2hrs at room temperature. Sera were diluted 30X followed by three-fold serial dilutions and incubated 2hr at room temperature 4°C on plates. Total IgG was detected with horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG (Invitrogen) diluted 1:32000. IgG1 was detected with HRP-conjugated goat anti-mouse IgG1 (Southern Biotech) diluted 1:16000. IgG2c was detected with HRP-conjugated rabbit anti-mouse IgG2c (Southern Biotech) diluted 1:5000. The enzyme reaction was developed with 3,3’,5,5’- tetramethylbenzidine, hydrogen peroxide (TMB Plus; Kementec), stopped with 0.2 M H2SO4 solution and plates read at 450 nm with 620 nm background correction using an ELISA reader (Tecan Sunrise).
The Mouse U-plex kit for the cytokines IL-12p70, IL-6, TNFα, MCP-1 was performed according to the manufacturer’s instructions (Meso Scale Discovery) to measure cytokine concentrations in serum. The plates were read on the Sector Imager 2400 system (Meso Scale Discovery) and calculation of cytokine concentrations in unknown samples was determined by 4-parameter logistic non-linear regression analysis of the standard curve.
3
Quantification of Inflammatory Markers and Anti-Collagen Antibodies
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The levels of TNF-α, IL-1β, IL-6, and BAFF in serum were measured with ELISA kits (R&D Systems Minnesota, USA) according to the manufacturer’s instructions.
In addition, serum levels of anti-CII antibodies were detected using an established ELISA. In brief, the wells of a 96-well plate (Dynatech Laboratories, Chantilly, USA) were coated with 500 ng of purified CII collagen (Sigma-Aldrich, Saint Louis, USA) in 100 μl PBS and incubated at 4 °C overnight. After being washed with PBST three times, the plate was blocked with 5% skimmed milk powder in PBST. The serum samples were obtained at day 42 after treatment and diluted to 1:10,000 and 1:100,000. The diluted serum samples were added to the plate, which was then incubated at 37 °C for 1 h. After washing the plate three times with PBST, total anti-CII antibody levels were detected with HRP-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL), and the IgG subclasses levels were determined using HRP-conjugated goat anti-mouse IgG1, IgG2a, or IgG2b (Southern Biotech, Birmingham, AL). Then, the substrate was developed with 50 μl of 3,3′,5,5′-tetramethylbenzidine (Southern Biotech, Birmingham, AL, USA) for 10 min, and the reaction was stopped with 50 μl of 0.5m H2SO4. The absorbance was measured at 450 nm and 570 nm with an ELISA reader (Thermo Scientific, Multiskan Mk3, USA).
In addition, serum levels of anti-CII antibodies were detected using an established ELISA. In brief, the wells of a 96-well plate (Dynatech Laboratories, Chantilly, USA) were coated with 500 ng of purified CII collagen (Sigma-Aldrich, Saint Louis, USA) in 100 μl PBS and incubated at 4 °C overnight. After being washed with PBST three times, the plate was blocked with 5% skimmed milk powder in PBST. The serum samples were obtained at day 42 after treatment and diluted to 1:10,000 and 1:100,000. The diluted serum samples were added to the plate, which was then incubated at 37 °C for 1 h. After washing the plate three times with PBST, total anti-CII antibody levels were detected with HRP-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL), and the IgG subclasses levels were determined using HRP-conjugated goat anti-mouse IgG1, IgG2a, or IgG2b (Southern Biotech, Birmingham, AL). Then, the substrate was developed with 50 μl of 3,3′,5,5′-tetramethylbenzidine (Southern Biotech, Birmingham, AL, USA) for 10 min, and the reaction was stopped with 50 μl of 0.5
4
Influenza Antibody and Neutralization Assays
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The procedures used to measure HA-specific antibodies were previously described53 (link). Briefly, 96-well plates (Nunc) were coated with 1 μg/mL HA protein (Influenza A H1N1 (A/Puerto Rico/8/1934) Haemagglutinin, SinoBiological) in Coating Buffer (0.1 M Na2CO3, 0.1 M NaHCO3, pH 9.5) at 4 °C overnight. HRP-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates), and HRP-conjugated anti-mouse IgG (R&D Systems) were used at 1:2000 to detect antigen-specific antibodies in serum. The procedures used in the microneutralization assay were previously described53 (link). Briefly, MDCK cells were seeded into 96-well plates on day –1. Then, these cells were washed twice with PBS and incubated in DMEM with 2 μg/mL trypsin (T1426, Sigma-Aldrich) at Day 0. Serum samples were serially diluted 2-fold in 50 μL of DMEM and then mixed with 100 TCID50 of PR8 influenza virus in 50 μL of DMEM for 1 h at 37 °C. 1 h later, the virus-serum mixture was transferred to MDCK cells and incubated for 24 h. After 24 h of incubation, the supernatant was removed, the cells were washed twice with PBS and fixed in 80% acetone for 30 min, and viral antigen was detected by ELISAs with a polyclonal antibody against NP protein. The OD450 was recorded. Viral plaque assays were performed as previously described57 (link).
5
NP5-BSA Splenocyte IgG1 Assay
6
Serological Assay for Nipah Virus Antibodies
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To determine the titer and subtype of specific antibodies against NiV in serum, we developed an indirect enzyme-linked immunosorbent assay (iELISA) [25 (link)]. In brief, 0.5 μg/mL of F or G proteins of NiV were coated onto ELISA plates overnight at 4 °C. After three washes with PBST, the plate was blocked with 3% bovine serum albumin (BSA) for 2 h. After three washes with PBST, 50 μL/well of the diluted inactivated serum samples were added and incubated for 1 h at 37 °C. After washing, a 1:10,000 dilution of HRP-conjugated goat anti-mouse IgG (CWBIO, Taizhou China), a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG1 (Southern Biotech, Birmingham, AL, USA), or a 1:5000 dilution of HRP-conjugated goat anti-mouse IgG2a (Southern Biotech, Birmingham, AL, USA) was added and incubated at 37 °C for 1 h. After TMB color development, the OD450nm value of each well was read. A test sample/negative sample with an OD450nm greater than 2 was considered positive.
7
MERS-CoV Neutralizing Antibody Assay
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Serum samples and nasal swabs were further tested for neutralizing antibodies against MERS-CoV (Qatar15/2015 and EMC/2012 isolates) using a plaque reduction neutralization (PRNT) assay. PRNT assay was carried out using according to the previously published protocol [19 (link)] with some modification. Briefly, samples were first inactivated at 56°C for 30 min. Then, 50 μl of 2-fold serial dilutions of heat-inactivated serum were mixed 1:1 with virus (400 PFU) prior to over-layering onto Huh7 cells. After 8 h of infection, the cells were fixed and stained using mouse anti-MERS-CoV nucleocapsid protein (SinoBiological) and HRP-conjugated goat anti-mouse IgG1 (SouthernBiotech). The number of infected cells were detected using a precipitate-forming TMB substrate (True Blue, KPL) and counted using an ImmunoSpot® Image analyser (CTL Europe GmbH). The PRNT titre was calculated based on a 50% or greater reduction in infected cells counts.
8
Western Blot Detection of PrPres
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Detection of PrPres by WB was performed as previously described (O'Rourke et al., 2011 (link), 2012 (link)). Briefly, 10 % brain homogenates in PBS were diluted by adding an equal volume of 2× lysis buffer (20 mM Tris-HCl pH 7.5; 1 % NP-40, 1 % sodium deoxycholate) and incubated with proteinase K (PK). Goat, sheep, WTD and TgElk brain homogenates were incubated with 50 µg ml−1 PK, whereas Tg338 brain homogenates were incubated with 100 µg ml−1 PK for 60 min at 37 °C. Sample loading buffer (2× NuPAGE LDS Sample Buffer, Invitrogen) was added prior to electrophoresis through 12 % Bis-Tris protein gels (Invitrogen) followed by transfer of proteins to PVDF membranes. Membranes were blocked with Blocker Casein (Pierce/Thermo Scientific) prior to incubation with the anti-prion antibody F99/97.6.1 (3.5 µg ml−1) (O'Rourke et al., 2000 (link)) and HRP-conjugated goat anti-mouse IgG1 (1 : 5000; Southern Biotechnology). Chemiluminescent signal of bound antibody (Amersham ECL; GE Healthcare) was captured on film (Carestream Health BioMax™ Light Film; Fisher Scientific).
9
Quantification of EV71-specific IgG Antibodies
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The EV71-specific IgG antibody response in mouse serum was determined by enzyme-linked immunosorbent assay (ELISA), as described previously [17 , 23 (link)], with slight modifications. The EV71 vaccine strain (100 ng/100 μL) diluted in coating buffer (0.1 M sodium carbonate) was used as a coating antigen in 96-well microplates (Corning Costar) at 4°C overnight. After blocking with 5% skim milk powder in PBS at 37°C for 2 h, 100 μL serum diluted 1:100 in dilution buffer (2.5% skim milk in PBS) was added to the microplates. After 2 h incubation at 25°C, 100 μL horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) antibody (Novex) diluted 1:2000 in dilution buffer was added, and samples were then incubated at 37°C for 1 h. After washing, 50 μL of 3,3’,5,5’-tetra-methylbenzidine (TMB) solution (Rockland) was added for development, followed by the addition of 100 μL of 2 M H2O4 to stop the reaction. Absorbance was measured at 450 nm using a microplate reader.
The profile of EV71-specific IgG isotypes was determined by ELISA using virus-coated plates, as described above. All protocols were similar, with the exception of the secondary antibodies, which were HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotech, Birmingham, AL).
The profile of EV71-specific IgG isotypes was determined by ELISA using virus-coated plates, as described above. All protocols were similar, with the exception of the secondary antibodies, which were HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotech, Birmingham, AL).
10
Characterization of Immune Cell Subsets
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H2O2 and CT were obtained from Sigma-Aldrich (St. Louis, MI, USA). Anti-mouse CD3-APC, CD4-FITC, CD8-PE, CD11c-eFluor 450, MHCII-APC, CD5-FITC and B220-PerCP-eFluor™ 710 antibodies were bought from eBioscience (San Diego, CA, USA). Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgA, HRP-conjugated goat anti-mouse IgG, HRP-conjugated goat anti-mouse IgG1 and HRP-conjugated goat anti-mouse IgG2a were obtained from Southern Biotech (Birmingham, AL, USA). RNA-easy Isolation was purchased from Vazyme Biotech Co., Ltd. (Nanjing, Jiangsu Province, China). SYBR qPCR Master Mix for quantitative real-time PCR (qRT-PCR) was bought from Yeasen Biotechnology Co., Ltd. (Shanghai, China). TMB was purchased from Beyotime Biotechnology (Shanghai, China). Red blood cell lysis buffer was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Silane coupling agent (KH550) and ZnONPs (purity 99.9%) were the products of Sinopharm Chemical Reagent Ltd. (Shanghai, China). The purified AMP was prepared in our laboratory (Yangzhou, Jiangsu Province, China). H2SO4, alcohol and HCl were obtained from Hushi Laboratory Equipment Co., Ltd. (Shanghai, China). Elisa plate was bought from cellpro biotechnology Co., Ltd. (Suzhou, Jiangsu Province, China).
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