Immulon 4HBX 96-well plates (Fisher, 14–245-153) were coated with 50 μL protein antigens at 2μg/ml in PBS and incubated overnight at 4°C. Plates were washed three times and blocked for one hour with 125 μL of blocking buffer (PBS with 3% goat serum, 0.5% nonfat milk, 0.1% Tween 20). Two-fold serial dilutions of serum samples were made in blocking buffer starting at a 1:50 dilution. Plates were washed three times, then diluted serum was added to plates and incubated for two hours. Detection antibodies conjugated to HRP were diluted in blocking buffer: rabbit anti-mouse IgM (Jackson Immunoresearch, 315–035-049) diluted 1:5000; goat anti-mouse IgK (Novus, NB7549) diluted 1:5,000; goat anti-mouse IgG1 (Southern Biotech, 1071–05) diluted 1:5,000; goat anti-mouse IgG2a (Southern Biotech, 1081–05) diluted 1:2,500. Plates were washed three times and diluted detection antibodies were added to plates and incubated for one hour. Plates were washed three times and 50μL of room temperature KPL SureBlue TMB substrate was added to wells. Plates incubated for 5 minutes before quenching with 25μL of 250mM HCl. Well absorbance was read at 450 nm. All wash steps were conducted using PBS with 0.1% Tween 20.
Immulon 4hbx 96 well plates
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Immulon 4HBX 96-well plates are a type of laboratory equipment designed for various scientific applications. They feature a high-binding surface, which provides efficient immobilization of proteins, peptides, and other biomolecules. The plates are made of polystyrene and are suitable for use in techniques such as enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and other applications where reliable binding of samples is required.
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Lab products found in correlation
Streptavidin hrp, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mouse igm, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse igg1, by Southern Biotech (1 mentions)
Goat anti mouse igg2a, by Southern Biotech (1 mentions)
Bovine serum albumin fraction 5, by Thermo Fisher Scientific (1 mentions)
Triturus elisa instrument, by Grifols (1 mentions)
Anti human igg horseradish peroxidase hrp conjugate, by Southern Biotech (1 mentions)
Synergy 4, by Agilent Technologies (1 mentions)
Goat anti mouse igg2b hrp, by Abcam (1 mentions)
Sigmafast opd tablet, by Merck Group (1 mentions)
Opteia mouse ige elisa kit, by BD (1 mentions)
Goat anti mouse igg2a hrp, by Abcam (1 mentions)
Sba clonotyping system c57bl 6 hrp, by Southern Biotech (1 mentions)
Monosialoganglioside gm1, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg, by MP Biomedicals (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Filtermax f5 plate reader, by Molecular Devices (1 mentions)
Omicron s, by Sino Biological (1 mentions)
Hrp conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
15 protocols using immulon 4hbx 96 well plates
1
ELISA Protocol for Antibody Detection
2
AIPL1 Variant Binding Assay
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Assays were performed as described in Sacristan-Reviriego et al.5 (link). Briefly, purified HSP90α or HSP90β (80 nM) were incubated in Immulon 4HBX 96-well plates (Fisher Scientific) for at least 1 h at 4 °C. 96-well plates were blocked using 1% blocking reagent (Sigma) in the same buffer (100 mM NaHCO3 pH 8.5) for 1 h and then washed 3 times with chilled TBST (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.075% Tween-20) to remove excess protein. Cell lysates from transfected cells with wild type (w/t) AIPL1 or AIPL1 variants were prepared as described above and added to the wells in lysis buffer (20 mM Tris HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.075% Tween-20) containing 2% protease inhibitor cocktail. After a 1 h incubation at 4 °C, wells were washed 5 times with chilled lysis buffer. Incubation with mouse anti-myc antibody (1:1000) for 1 h was followed by 5 washes and another 1 h incubation with anti-mouse HRP conjugated antibody (1:10,000). After 5 final washes, the plate was incubated with substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma) for colorimetric detection at 30 °C for 30 min. The reaction was stopped using 0.5 M H2SO4 and the absorbance measured at 450 nm. For comparison of the AIPL1 variants to w/t AIPL1, the absorbance measured at 450 nm was normalised to the expression level in cell lysates detected by SDS-PAGE and western blotting.
3
Quantifying NP-specific AFC Response
4
Quantification of Soluble TNF-α by ELISA
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TNF-α concentrations were determined using a commercially available ELISA antibody pair kit (Life Technologies, Camarillo, CA). Briefly, Immulon 4HBX 96-well plates (Thermo Scientific, Waltham, MA) were coated with 2 μg mL−1 capture antibody in the DPBS buffer (137.0 mM NaCl, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, 0.1% ProClin™, pH = 7.4) over night at 4 °C. Plates were washed and then blocked with the DPBS buffer containing 0.1% (v/v) Tween-20 and 0.5% bovine serum albumin (BSA) fraction V (Fisher Scientific, Fair Lawn, NJ) for 1 hr. Microtiter plates were then loaded with recombinant human TNF-α samples spiked in the DPBS buffer and incubated at room temperature for 2 hr with 0.32 μg mL−1 detection antibody. Following incubation, microtiter plates were washed and then incubated with streptavidin-horseradish peroxidase (HRP) conjugates for 30 min at room temperature. Microtiter plates were washed and incubated with TMB stabilized chromagen solution (Life Technologies, Camarillo, CA) for 20 min, before the reaction was stopped using 1.8N H2SO4 solution. Absorbance of each well at a wavelength of 450 nm was measured using the M3 SpectraMax multi-mode plate reader (Molecular Devices, Sunnyvale, CA).
5
Measuring Human IgG Binding to PF4
6
IgG Binding Assay for PF4 Mutants
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Binding of human IgG was measured essentially as previously described for KKO and RTO antibodies7 (link). Briefly, Immulon 4 HBX 96-well plates (Thermo Fisher Scientific, Waltham, MA) were coated overnight with either PF4 or PF4 mutant at 5 ug ml−1. The plates were incubated for 30 min with either PBS (control) or with 0.5% glutaraldehyde at room temperature, extensively washed and blocked with 1% bovine serum albumin (BSA) in PBS. The plates were incubated with human patient IgG samples at experimentally selected concentration of 20 μg ml–1 for 30 min at 37 °C. IgG binding was measured as absorbance at 405 nm (A405) after incubation for 30 min at 37 °C with horseradish peroxidase-conjugated ImmunoPure Goat Anti-Human IgG (H+L), HRP Conjugated Product No. 31412 (Pierce. Rockford, IL) diluted 1:10,000 in 1% BSA/PBS. Horseradish peroxidase substrate ABTS was from Roche Applied Science, Penzberg, Germany. Absorbance was measured with a SpectraCount plate reader (Packard BioScience, Waltham, MA).
7
Epitope Profiling Immunocapture Competition Assays
8
Serological Diagnosis of M. pneumoniae
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Diagnostic of M. pneumoniae was conducted using the Liaison M. pneumoniae IgG, IgM kit (DiaSorin) using a 1/100 dilution of the patient sera. Indirect ELISA assays were performed on 96-well plates Immulon 4 HBX 96-well plates (ThermoFisher) incubating 1 µg of each antigen at 4 °C overnight. 1/100 dilutions of each patient serum were added to the plate and detected using an anti-human IgG antibody conjugated with HRP (Thermofisher Scientific). Upon incubation for 30 min with 100 µl of substrate (Thermofisher Scientific), 100 µl of sulfuric acid 25% was added to stop the reaction and absorbance was read at 450 nm on a Triturus ELISA instrument (Grifols) device. Reference filter was set at 620 nm.
9
Peptide-binding ELISA for Antibody Characterization
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Immulon 4HBx 96-well plates (Thermo Fisher, Waltham, MA) were coated overnight with 100 ng/well of the following peptides at 4 °C: full-length FL-CSP, NANPx6, peptide 21 (NPDPNANPNVDPNAN)12 (link), or NPDP19 peptide (KQPADGNPDPNANPNVDPN)21 (link). Plates were washed thrice with PBS-Tween (1X PBS + 0.05% tween) solution and blocked for 1.5 h at room temperature with 0.5% casein + 1% Tween solution. MAbs were plated starting between 1000 and 200 ng/ml and serially diluted threefold down the plate. After 2 h incubation at room temperature, unbound antibodies were washed away thrice with PBS-Tween solution and remaining mAbs were detected using Anti-human IgG-horseradish peroxidase (HRP) conjugate (1:5000; Southern Biotech, Birmingham, AL) as secondary antibody along with 100 µl per well of ABTS 2-component substrate (KPL, Gaithersburg, MD) for color development for 1 h. After stopping the reaction with 20% SDS, optical density at 415 nm (OD415) was measured on a Synergy 4 microplate reader (BioTek, Winooski, VT). Concentration of mAb needed to achieve an OD of 1 using Gen5 software (BioTek, Winooski, VT).
10
Epitope Profiling Competition Assay
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Epitope profiling immunocapture competition (EPICC) assays were conducted as described (20 (link)). Clear, flat-bottom Immulon 4HBX 96-well plates (Thermo Fisher Scientific, Pittsburgh, PA) were coated overnight with a capture anti-RTA MAb (1 μg/ml). The following day, the plates were blocked with 2% goat serum, washed, and then overlaid with a mixture of biotin-tagged RT and polyclonal antibody (pAb) mouse serum (1:25). The amount of biotin-RT used in the competition ELISA was equivalent to the 90% effective concentration (EC90) for each capture MAb (range, 100 to 150 ng/ml). After 1 h of incubation, plates were washed and developed with streptavidin-HRP (1:1,000; Thermo Fisher Scientific, Pittsburgh, PA) and TMB, as described above for ELISAs. The percent inhibition of RT binding to the capture MAb in the presence of pAb serum was calculated from the optical density (OD) values as follows: 1 − value OD450 value (biotin-RT + competitor MAb)/OD450 (biotin-RT without competitor MAb) × 100.
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