- AU5 (See other available formats)
- Other Names
- TDFYLK epitope tag, TDFYLK tag
Covance Catalog# AFC-135P
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The AU5 tag, which consists of the peptide sequence TDFYLK, is often used as a protein modification in order to simplify the labeling and detection of proteins. This unique amino acid sequence allows for specific antibody detection in western blotting and other immunostaining techniques. Due to the short sequence, this modification is not likely to affect the structure or function of the modified proteins.Product Details
- TDFYLK Tag
- Antibody Type
- The AU5 monoclonal antibody was raised against an SDS-disrupted BPV-1 preparation and mapped to the six amino acid epitope TDFYLK.
- Purified IgG immobilized on Sepharose™ Fast Flow beads (in PBS + 0.03% Thimerosal).
- The antibody was purified by affinity chromatography.
- Storage & Handling
- Store between 2-8°C. The matrix may be re-used several times. To strip column after use, wash with several bead volumes of 0.1 M glycine pH 2.8 followed immediately by PBS containing 0.3% Thimerosal or 1mM Sodium Azide as preservative.
- Recommended Usage
Each lot of this antibody is quality control tested by immunoprecipitation.
The optimal buffers and matrix concentration should be determined for each specific assay condition.
Binding: Tagged protein will bind to matrix in common physiologic buffers with pH in the range of 6.0-7.5, salt from 50-500 mM and in the presence of reasonable levels of detergent. Excess reducing agent should be avoided as the disulfide bridges holding antibody heavy and light chains may be compromised. BioLegend tests Affinity Matrix using an equilibration/binding buffer containing:
• 100 mM Tris-HCl (pH 7.5)
• 150 mM NaCl
• 0.1% Tween 20
• 0.5% BSA
• 1 mM beta-mercaptoethanol
Washing: After binding, washes with several bead volumes of buffer are recommended. Such buffer may contain increased salt, altered pH, etc. as determined empirically to remove un-tagged proteins.
Elution: Several options are available for elution.
1. SDS gel loading buffer may be applied directly to the beads in order to display all bound protein on a polyacrylamide gel/western. Note that gel loading buffer containing reducing agent will also release some antibody heavy and light chains (approx 25 and 50kD, respectively).
2. Competitive elution with epitope peptide. For epitope tag affinity matrices, prepare an elution buffer with epitope tag peptide at 400 ug/mL in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA (pH 8.0).
3. Chemical Elution. Elution by pH or chaotropic salts is also possible. For elution by pH, either 0.1 M glycine pH 2.8 or 40 mM diethyl-amine pH 11.0 may be used.
- Application Notes
This affinity matrix can be used for immunopurification of AU5 tagged fusion proteins from crude starting material. On an analytical scale, it can also be used for immunoprecipitating AU5 tagged proteins.
Sepharose is a trademark of Amersham Biosciences Limited
Monoclonal mouse antibody AU5 recognizes the peptide epitope, TDFYLK. This antibody was purified using protein-G chromatography, and was subsequently immobilized onto a Sepharose™ fast flow matrix.
- Application References
- Field J, et al. 1988. Mol Cell Biol. 8:2159.
AB_2564998 (BioLegend Cat. No. 900701)
- Biology Area
- Cell Biology
- Gene ID
- View information about AU5 on UniProt.org
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