Recombinant HRP Anti-Kappa light chain antibody [EPR5367-8] (ab202549)

Ab202549 was tested using a sandwich ELISA approach. The wells were coated with ab195576 at 2µg/ml at 50µl/well overnight at 4°C, followed by a 5% BSA blocking step for 2h RT. Native Human Kappa Light Chain (Biorad) was then added starting at 40 µg/ml and plasma/serum at 1:200 and gradually diluted 1:4, 50µl/well for 2h. Ab202549 was then added at 1:10,000 dilution, 50µl/well for 2h.

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab202549 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

IHC image of human kappa light chain staining in a section of formalin-fixed paraffin-embedded normal human tonsil*, performed on a Leica Bond system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab202549, at 1/100 dilution, for 15 mins at room temperature and detected using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Staining corresponds to B cells expressing IgG commonly found in lymphoid follicles of human tonsil.

The inset background control image is taken from an identical assay without added antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Negative IHC image of human kappa light chain staining in a section of formalin-fixed paraffin-embedded normal human cerebral cortex*, performed on a Leica Bond system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab202549, at 1/50 dilution, for 15 mins at room temperature and detected using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Staining corresponds to immunoglobulins found in serum. As expected, staining diminishes further into the brain tissue due to the action of the blood-brain barrier.

The inset background control image is taken from an identical assay without added antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre