To determine the presence of epitope-tagged antibodies in IgkTag mice, serum samples from steady state adult mice and precision plus dual color protein standards (Biorad) were run in triplicate on SDS-PAGE mini-protean TGX protein gels (Biorad) under denaturing conditions, to separate heavy and light antibody chains. Samples were transferred to a PVDF membrane using the Iblot gel transfer system (Invitrogen). Membranes were blocked for 2 hours at room temperature while gently shaking with 5% nonfat dry milk in PBS-Tween (0.05%), prior to overnight incubation in the same buffer with 1:2000 anti-FLAG-HRP (clone D6W5B, CellSignallingTechnology #86861S) or anti-Strep (clone Strep-tag II StrepMAB-Classic, Biorad #MCA2489P) or goat anti-mouse Igκ-HRP (SouthernBiotech #1050-05). Membranes were extensively washed with PBS-Tween and subsequently incubated with western blotting ECL substrate (Amersham) prior to chemiluminescence detection on an Azure c300 gel imager (Azure Biosystems).
Goat anti mouse igκ hrp
Manufactured by Southern Biotech
Goat anti-mouse Igκ-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse kappa light chain immunoglobulins (Igκ) in various immunoassays and other applications.
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Lab products found in correlation
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Iblot gel transfer system, by Thermo Fisher Scientific (1 mentions)
Azure c300 gel imager, by Azure Biosystems (1 mentions)
Sba clonotyping system horseradish peroxidase hrp kit, by Southern Biotech (1 mentions)
Clonotyping system hrp kit, by Southern Biotech (1 mentions)
Immunospot analyzer, by Cellular Technology (1 mentions)
Goat anti mouse igg1 hrp, by Southern Biotech (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
2 protocols using goat anti mouse igκ hrp
1
Antibody Detection in Igk^Tag Mice
2
Quantification of Isotype-Specific NP-Reactive Antibodies
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Total levels of immunoglobulins of different isotypes in the sera or culture supernatants were quantified using SBA Clonotyping system-horseradish peroxidase (HRP) kit (Southern Biotech, cat # 5300-05) according to the manufacturer’s recommendation. NP-specific IgM, IgG1, IgG2a, IgG2b, and IgG3 in the serum was measured using plates (Maxisorp, ThermoFisher, cat # 439454) coated with NP20-BSA (Bioresearch Technologies, cat # N-5050H) and Clonotyping system-HRP kit (Southern Biotech). Pooled sera from NP-KLH-immunized WT mice, 14 days post immunization were used as standard control. ELISpot assays were performed for the detection of NP-specific ASCs. Multiscreen membrane filtration 96-well plates (EMD Millipore, cat # MAIPSWU10) were activated by 35% ethanol and coated with 25 µg/ml NP-BSA. Cells from spleen and bone marrow were seeded into wells with and incubated overnight at 37 °C. Wells were washed and developed using goat anti-mouse IgG1-HRP or goat anti-mouse Igκ-HRP (Southern Biotech, cat # 5300-05, 1:1000) and 3,3′-diaminobenzidine (Vectorlabs, cat # SK-4100). The spots were counted manually and photographed using ImmunoSpot analyzer (Cellular Technology Limited).
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