96-well microplates (Greiner Bio-one, Kremsmünster, Austria) were coated with 100 μL of an appropriate dilution of RVG-VLPs in carbonate buffer pH 9.6 for 1 h at 37 °C and overnight at 4 °C. Plates were washed six times with PBS-Tween-20 0.05% and then blocked with 200 μL per well of 2% skim milk in PBS for 1 h at 37 °C. Two-fold serial dilutions of sera samples in skim milk 0.2%-PBS Tween-20 0.05% were incubated for 1 h at 37 °C. After that, 100 μL of an HRP conjugated anti-mouse antibody (Dako, Agilent Technologies, Santa Clara, CA, USA) diluted 1:1000 in skim milk 0.2%-PBS tween-20 0.05% was added to each well and incubated for 1 h at 37 °C. A total of six washes with PBS-Tween--20 0.05% were done between each incubation. Finally, the reaction was revealed adding 100 μL of a chromogenic substrate solution to each well (0.5 mg.mL−1 o-phenylenediamine (Sigma-Aldrich, Saint Louis, MI, USA), 0.5 μL/mL H2O2 30 vol., 50 mM citrate/phosphate buffer, pH 5.3). The reaction was stopped adding 50 μL of a 0.5 M H2SO4 solution and the optical density was measured at 492 nm in a plate reader spectrophotometer (Labsystems Multiskan®). Antibody titers were calculated as the end-point serum dilution yielding an optical density higher than the cut-off value. This cut-off was calculated as the mean + 2 S.D. of the optical density of negative controls (basal mice sera).
Hrp conjugated anti mouse antibody
Manufactured by Agilent Technologies
Sourced in United States
The HRP-conjugated anti-mouse antibody is a laboratory reagent used for the detection and quantification of mouse-derived proteins in various assays. It consists of a mouse-specific antibody that is conjugated to the enzyme horseradish peroxidase (HRP). This conjugation allows for the amplification of signal when the antibody binds to its target, enabling sensitive and reliable detection.
Automatically generated - may contain errors
Lab products found in correlation
O phenylenediamine, by Merck Group (1 mentions)
96 well microplate, by Greiner (1 mentions)
T6557, by Merck Group (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Sc 393123, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Swine anti rabbit hrp conjugated antibody, by Agilent Technologies (1 mentions)
Anti tetra his antibody, by Qiagen (1 mentions)
Rabbit anti human fibrinogen antibody, by Agilent Technologies (1 mentions)
7 protocols using hrp conjugated anti mouse antibody
1
ELISA for RVG-VLP Antibodies
2
Immunoblotting with Specific Antibodies
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Primary antibodies used in this study include antibodies to EHHADH (sc-393123, Santa Cruz Biotechnology, 1:500 dilution), VHL (68547, Cell Signaling Technology, 1:1000 dilution), and γ-tubulin (T6557, Sigma-Aldrich, 1:4000 dilution). Secondary antibodies used include HRP-conjugated anti-rabbit (7074, Cell Signaling Technology, 1:5000 dilution) and HRP-conjugated anti-mouse antibody (P0447, Dako, Agilent Technologies, 1:10000 dilution).
3
Western Blot Analysis of MYCN and MAX
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Whole cell extracts were prepared and lysed using RIPA-buffer. Western blot analysis was performed as previously described [54] (link). In brief, membranes were probed with a mouse anti-MYCN antibody (B8.4.B, St Cruz Bio), MAX antibody (C-2, St Cruz Bio) and an HRP-conjugated anti-mouse antibody (DAKO) was used as secondary antibody. The membranes were developed using enhanced chemiluminescence substrate (ECL, Amersham). The membranes were subsequently re-probed with antibodies specific for GAPDH (6C5, St Cruz Bio) in order to confirm equal loading.
4
KSHV Lytic Reactivation Inhibition
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Cultured RPE‐1 cells were infected with KSHVLYT (MOI = 0.07) and treated with hesperidin, eupafolin, or foscarnet. After incubation for 5 days, the cells were lysed in RIPA buffer (50 mm Tris‐HCl, pH 7.4; 150 mm NaCl; 1% NP‐40; 0.25% sodium deoxycholate; 1 mm EDTA), supplemented with protease inhibitors (Roche, Ascur Versicherungsvermittlungs GmbH, Grenzach‐Wyhlen, Germany). The mixture was subjected to SDS‐PAGE before transferring separated electrophoresis products to a nitrocellulose membrane. Mouse monoclonal antibodies anti‐ORF45, anti‐K8.1A/B (Santa Cruz Biotechnology, Dallas, TX, USA), and anti‐β‐actin (Sigma‐Aldrich) were used to detect respective proteins after adding HRP‐conjugated antimouse antibody (Dako, Santa Clara, CA, USA) as secondary antibody. Blots were scanned using Image Scanner, and a densitometric quantification was performed using image j freeware. The mean relative density for each target band was normalized against the density of β‐actin.
5
HBsAg-HCVcp Fusion Protein Expression Analysis
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Expression of the HBsAg-HCVcp fusion protein by pCHCORE was assayed in transiently transfected HEK 293T cells by western blotting analysis. In brief, cells were transfected with pCHCORE using lipofectamine (GIBCO-BRL, Scotland). Subsequently, transfected cells were resuspended in lysis buffer, containing 0.1 M Tris–Cl (pH 7.8) and 0.5% (V/V) Triton X-100. Cell lysate was used to detect HBsAg-core fusion protein using primary mouse anti-HBsAg polyclonal antibody followed by incubation with HRP-conjugated anti-mouse antibody (DAKO, Denmark). Accordingly, purified recombinant HCVcp was analyzed by SDS-PAGE and western-blot analysis as previously described.[20 (link)22 (link)23 (link)]
6
Immune Capture ELISA for Trypanosoma Antigens
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The Immune capture ELISA was assayed in 96 well plates MaxiSorp (Nunc, Thermo ScientificTM) coated with a 4 μg/ml of anti- MASP SP rat immune sera in a volume of 100 μl/well and diluted in (0.1 M) bicarbonate-carbonate buffer (pH 9.6), overnight at 4 °C. After 3X washes with PBST, 300 μl/well of blocking buffer (skimmed milk 2% (V/W) in PBS-T) was added for 1 h at RT. Following 3X washes in PBST, 5 μg of ICs-free total protein from the different positive sera (CARD-Arr, CARD-ICC, DIG and ASYMP) were added in a final volume of 50 μl in PBS and incubated overnight at 37 °C (or EVs obtained from the Trypomastigote cultures depending of the experiments). After 3X washes with PBS and depending on the assay, the wells were incubated for 1 h at 37 °C with 5 μg/ml per well of anti-EVs-Trypo mouse immune sera, anti-TPTC rabbit immune sera, anti-CD9 antibody mouse immune sera (Biolegend), a dilution of positive sera (1:100) without ICs or PBS as negative control. After 3 washes with PBS, the plate was incubated with 100 μl of HRP-conjugated anti-mouse antibody (DAKO), HRP-anti-rabbit antibody (DAKO) or HRP-conjugated IgG anti-human (Sigma) as secondary antibodies at a dilution 1∶50000, 1:1000, 1:2000 and 1:1000 respectively in PBS for 1 h at RT. After the final 3 washes with PBS-T, the reaction was developed as described above.
7
Quantifying Protein-ECM Interactions
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ECM proteins in coating buffer (50 μl) were added to wells of a high‐binding 96‐well plate (Immulon 2HB) and incubated for 16 hr at 4°C. Wells were washed once in TBSC and non‐specific binding sites were blocked with 3% BSA in TBSC for 1 hr at 37°C. Wells were washed once in TBSC and recombinant protein (0–5 μg) diluted in TBSC was applied to the wells and incubated for 1 hr at 37°C. Unbound protein was removed and wells washed once in TBS. Primary antibody diluted in TBST was added to the wells and incubated for 1 hr at 37°C. Wells were washed twice in TBST before adding HRP‐linked secondary antibody diluted in TBST containing 3% BSA and incubating for 1 hr at 37°C. Wells were washed once in TBST, twice in TBS, and detection reagent (0.102 M Na2HPO4, 0.049 M citric acid, 0.012% H2O2, 3.7 mM o‐phenylenediamine) was added to wells. Plates were incubated in the dark for 10 min at room temperature, 0.56 M H2SO4 was added to stop the reactions and A490 measured. x6His‐tagged proteins were detected using anti‐tetraHis antibody (Qiagen) at 1:1000 dilution and HRP‐conjugated anti‐mouse antibody (Dako) at 1:2000 dilution. Fibrinogen was detected using rabbit anti‐human fibrinogen antibody (Dako) at 1:1000 dilution and HRP‐conjugated swine anti‐rabbit antibody (Dako) at 1:2000 dilution.
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