For immunoblotting, cells were washed with PBS and lysed in RIPA buffer with added protease and phosphatase inhibitors (Roche). Samples were sonicated, diluted in Laemmli sample buffer and separated by SDS‐PAGE before transferred to the PVDF membrane. Membranes were probed with antibodies listed in Table S1 and a secondary antibody conjugated to horseradish peroxidase, before developing using enhanced chemiluminescence substrate. For IFN‐I ELISA, 96 well plates were coated with a mixture of antibodies against IFN‐α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS. Samples along with recombinant IFN‐α2 (Miltenyi), for construction of the standard curve, were added in triplicate and incubated overnight at 4°C. The plate was washed and IFN‐I assayed using a mixture of biotinylated detection antibodies against IFN‐α (Mabtech, MT2/4/6). Avidin‐conjugated alkaline phosphatase (Sigma, ExtrAvidin), and p‐Nitrophenyl phosphate substrate (Sigma, SigmaFast tablets) were used to develop the ELISA. Absorbance was read at 405 nm on a Multiskan EX plate reader (Thermo Fisher).
Mt1 3 5
Manufactured by Mabtech
The MT1/3/5 is a versatile piece of lab equipment used for the detection and quantification of cytokines and other analytes in biological samples. It is designed to provide accurate and reliable results through the use of a sandwich ELISA format. The core function of the MT1/3/5 is to facilitate the measurement of target analytes in a controlled and standardized manner.
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2 protocols using mt1 3 5
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Immunoblotting and IFN-I ELISA Protocols
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Immunoblotting and ELISA Techniques
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Immunoblotting and ELISA assays: For immunoblotting, cells were washed with PBS and lysed in RIPA buffer with added protease and phosphatase inhibitors (Roche). Samples were sonicated, diluted in Laemmli sample buffer and separated by SDS-PAGE, before transfer to PVDF membrane. Membranes were probed with antibodies listed in Supplementary Table 1 and a secondary antibody conjugated to horseradish peroxidase, before developing using enhanced chemiluminesence substrate. For IFN-I ELISA, 96 well plates were coated with a mixture of antibodies against IFN-α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS. Samples along with recombinant IFN-α2 (Miltenyi), for construction of the standard curve, were added in triplicate and incubated overnight at 4°C. The plate was washed and IFN-I assayed using a mixture of biotinylated detection antibodies against IFN-α (Mabtech, MT2/4/6). Avidin-conjugated alkaline phosphatase (Sigma, ExtrAvidin), and p-Nitrophenyl phosphate substrate (Sigma, SigmaFast tablets) were used to develop the ELISA. Absorbance was read at 405nm on a Multiskan EX plate reader (Thermo Fisher).
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