Description
General description
Anti-HA-Biotin, High Affinity (3F10) is a monoclonal antibody for the highly sensitive detection of HA-tagged recombinant proteins, Fab fragments, conjugated to biotin. The Anti-HA-Biotin, High Affinity antibody (clone 3F10) recognizes the same epitope as clone 12CA5. It is a monoclonal antibody whose high affinity and low working concentrations result in less cross-reactivity than with other antibodies to the HA-epitope. Anti-HA-Biotin, High Affinity (3F10) is a biotin conjugate of this clone which is specifically useful in western blotting, ELISA applications and assays using the universal biotin-streptavidin platform, by allowing specific and highly sensitive detection of HA-tagged proteins.
Immunogen
Amino acids 98-106 from the human influenza virus hemagglutinin protein
Application
Anti-HA-Biotin, High Affinity (3F10) is used for the detection of HA-tagged recombinant proteins using:
Dot blots
ELISA (enzyme-linked immunosorbent assay)
Western blots
Since Anti-HA High Affinity is a rat monoclonal, it is possible to use it in conjunction with murine monoclonals for double labeling.
It has also been used for immunocytochemistry, immunofluorescence and αScreen format based assay.
Biochem/physiol Actions
Anti-HA-Biotin, High Affinity (3F10) recognizes the 9-amino acid sequence YPYDVPDYA, derived from the human influenza hemagglutinin (HA) protein. This epitope is also recognized in fusion proteins regardless of its position (N-terminal, C-terminal or internal).
Preparation Note
Sample Materials
Sample preparation: Prepare protein extracts containing the HA-tagged protein of interest using any of a variety of standard methods. The following lysis buffers have performed well and should be taken as guidelines:
Bacterial extracts: 20 mM Tris, pH 8.0, 100 mM NaCl, cOmplete Protease Inhibitor Cocktail Tablets, followed by freeze-thaw.
Mammalian extracts: 50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Nonidet P40, complete Protease Inhibitor Cocktail Tablets.
Other cell lysis buffers may be more appropriate for individual applications. In general, to obtain optimal performance of the affinity matrix:
Use protease inhibitors to reduce proteolytic activity. Use complete Protease Inhibitor Cocktail Tablets for most applications.
Limit detergent to the lowest concentration levels necessary to obtain adequate cell lysis.
Working concentration: Working concentration of conjugate depends on application and substrate
The following concentrations should be taken as a guideline:Dot blot: 100 ng/ml
ELISA: 100 ng/ml
Western blot: 100 ng/ml
Add 1 ml double-distilled water to a final concentration of 50 μg/ml.
Rehydrate for 10 minutes prior to use.
Analysis Note
Function test: The Anti-HA-Biotin; High Affinity is function tested by Western blot analysis of a HA-tagged fusion protein.
Other Notes
For life science research only. Not for use in diagnostic procedures.
