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Our Abpromise guarantee covers the use of ab28484 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 2 µg/ml. Detects a band of approximately 280 kDa (predicted molecular weight: 156 kDa).|
|ICC/IF||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 4 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
ab28484 (4µg/ml) staining lubricin in Human liver using an automated system. Using this protocol there is staining of the cytoplasm and nuclei in hepatic arteries and plates of hepatic cells.
Sections were rehydrated and antigen retrieved with buffers EDTA pH 9.0 . Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
Samples from normal bovine (NL bovine) and human osteoarthritic synovial fluids (OA human). The lubricin was purified by CsCl gradient and DEAE sepharose chromatography prior electrophoresis. ab28484 was used at a concentration of 2μg/ml
ab28484 works best with non-reduced samples as it looses a lot of its binding capapcity if the lubricin is reduced prior to SDS PAGE. Western blot analysis is difficult unless the protein sample is a recominant or purified sample.
The wide band, higher than expected molecular weight seen on the blot, are consistent with the high carbohydrate content of the protein sample.
For permeabilization, 0.25% Triton X-100 was included in primary and secondary antibody incubation buffers.
Cells were also stained with FITC-labeled Peanut Agglutinin. PNA binds lubricin and certain other glycoproteins, so some co-localization was expected between the Alexa Fluor 568 signal and the FITC signal.
Unexpected co-localization was noted between the Alexa Fluor 568 staining (indirect lubricin staining) and DAPI (nuclear counterstain) in the cell nuclei.
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