ELISAs were performed on 96-well half-area microplates (ThermoFisher Scientific) as described previously 66 (link). The plate was coated with 2 μg/mL mouse anti-His antibody (Invitrogen cat. #MA1-21315-1MG, ThermoFisher Scientific) overnight at 4°C. The following day, plates were washed three times with PBST (PBS + 0.05% Tween20) and incubated for 1h with blocking buffer (3% bovine serum albumin (BSA)). Following removal of blocking buffer, plates were treated with His-tagged proteins (5 μg/mL in PBST + 1% BSA) for 1.5h at room temperature. Plates were washed and serum was added at threefold dilutions (beginning at 1:30) and allowed to incubate for 1.5h. Following washes, secondary antibody (AffiniPure Goat anti-human IgG Fc fragment specific, Jackson ImmunoResearch Laboratories cat. #109-055-008) was added at 1:1000 for an additional 1h. Secondary antibody was washed, and staining substrate (alkaline phosphatase substrate pNPP tablets, Sigma) was added. Absorbance at 405 nm was measured after 8, 20, and 30 min using VersaMax microplate reader (Molecular Devices) and analyzed using SoftMax version 5.4 (Molecular Devices).
Softmax version 5
Manufactured by Molecular Devices
SoftMax version 5.4 is a software package designed to control and analyze data from a variety of Molecular Devices' laboratory equipment. It provides the core functionality to operate the equipment and collect experimental data.
Automatically generated - may contain errors
Lab products found in correlation
96 well half area microplates, by Thermo Fisher Scientific (2 mentions)
Ma1 21315 1mg, by Thermo Fisher Scientific (2 mentions)
Alkaline phosphatase substrate pnpp tablets, by Merck Group (2 mentions)
Versamax microplate reader, by Molecular Devices (2 mentions)
Affinipure goat anti human igg fc fragment specific, by Jackson ImmunoResearch (2 mentions)
2 protocols using softmax version 5
1
ELISA for His-tagged Protein Detection
2
ELISA-based Protein Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
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ELISAs were performed on 96-well half-area microplates (ThermoFisher Scientific) as described previously15 (link). The plate was coated with 2 μg/mL mouse anti-His antibody (Invitrogen cat. #MA1-21315-1MG, ThermoFisher Scientific) overnight at 4°C. The following day, plates were washed three times with PBST (PBS + 0.05% Tween20) and incubated for 1h with blocking buffer (3% bovine serum albumin (BSA)). Following removal of blocking buffer, plates were treated with His-tagged proteins (5 μg/mL in PBST + 1% BSA) for 1.5h at room temperature. Plates were washed and serum was added at threefold dilutions (beginning at 1:30) and allowed to incubate for 1.5h. Following washes, secondary antibody (AffiniPure Goat anti-human IgG Fc fragment specific, Jackson ImmunoResearch Laboratories cat. #109-055-008) was added for an additional 1h. Secondary antibody was washed, and staining substrate (alkaline phosphatase substrate pNPP tablets, Sigma) was added. Absorbance at 405 nm was measured after 8, 20, and 30 min using VersaMax microplate reader (Molecular Devices) and analyzed using SoftMax version 5.4 (Molecular Devices).
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