Ascities fluid obtained from mice immunized with a hybridoma-(clone BA4) producing antibody to bovine alpha-elastin. The epitope has been mapped to VGVAPG repeat in elastin. The antibody type is IgGI and reacts with protein A.
FORM and STORAGE.
Lyophilized. Store at 4°C until rehrdrated. Stable for a minimum of 1 year at 4°C. No preservatives added. Additional biological protection may be provided with 0.01% thimerosal. Non-sterile.
Rehydration and Storage:
Dilute to the volume indicated on the label with deionized water. Dilution with 50% glycerol, however, is preferred. This procedure eliminates the need for freezing, since 50% glycerol is an effective biological inhibitor when the product is stored at 4°C. At most working dilutions, the level of glycerol is too small to influence most assays.
Note: Variations in temperature due to freeze-thaw cycles may cause loss of activity when rehydrated product is stored in aliquots less than 50µl.
Tested by immunohistology as well as ELISA and Western blot techniques, this product reacts most strongly with bovine tropoelastin and insoluble forms of elastin. It does show cross reactivity with elastin from other species, although reactivity is somewhat lower for non-bovine elastins. Those elastins for which reactivity has been documented include human, rabbit, pig, goat, cat, guinea and sheep. This antibody shows little reactivity toward rat, mouse, or chicken elastins.
SUGGESTED WORKING DILUTIONS.
Different assay conditions require that serial dilution of all reagents be performed to determine optimal working concentrations. Prepare working dilution immediately before use. Storage of working dilution may result in performance loss. Suggesting starting dilutions are as follows:
- Microwell Plate Assays 1:1000 � 1:2000
- Western blots 1:200 1:500
- Immunoprecipitation 1:100 � 1:200
- Histo/Cytochemistry 1:50 � 1:100
ETCHING to ENHANCE ANTIGENICITY
In many cases, antigenicity of elastic fiber components can be enhanced by treating with 6 M guanidine HCl, 50 mM dithiothreitol, 20 mM Tris pH 8.0 for 15 minutes. After washing with 20 mM Tris pH 8.0, the slides are treated with 100 mM iodoacetamide in the dark for 15 minutes, washed and stained using normal protocols.