Description		Affinity Matrix: Monoclonal Mouse Antibody, HA.11
Intended Use		**Research Use Only (RUO)** This product is sold for laboratory research use only, not for human or in-vivo use.
Clone		16B12
Form		Purified IgG immobilized on Sepharose™ Fast Flow beads (in PBS + 0.03% Thimerosal)
[Ab]		2 mg/mL
Specificity		Monoclonal mouse antibody HA.11 recognizes the peptide epitope, YPYDVPDYA. This second-generation HA antibody is an excellent substitute for the 12CA5 monoclonal antibody. The HA.11 antibody recognizes HA epitopes located in the middle of protein sequences as well as at the N- or C-terminus. HA.11 antibody was purified using protein-G chromatography and was subsequently immobilized onto a Sepharose™ Fast Flow matrix.
Uses		This affinity matrix can be used for immunopurification of HA-tagged fusion proteins from crude starting material. On an analytical scale, it can also be used for immunoprecipitating HA-tagged proteins.
Suggested Working Dilution		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. Covance 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.
Notes		Sepharose is a trademark of Amersham Biosciences Limited
Storage		The matrix may be re-used several times. To strip column after use, wash with several bead volumes 0.1 M glycine pH 2.8 followed immediately by PBS cotaining 0.3% thimerosol or 1mM azide as preservative. Store at 4°C. This shelf-life of this product is approximately 1 year.
References		Ferrando A, Koncz-Kalman Z, Farras R, Tiburcio A, Schell J, Koncz C. Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells. Nucleic Acids Res 29(17):3685-93, 2001. J Field, J Nikawa, D Broek, B MacDonald, L Rodgers, I Wilson, R Lerner, M Wigler. Purification of RAS responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8:2159-2165, 1988.
Warranty/Conditions		Covance products may not be resold or modified for resale without prior written approval.