- 6-His (See other available formats)
- Other Names
- 6-His Epitope Tag
Covance Catalog# AFC-156P
- Mouse IgG1
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Monoclonal mouse antibody 6-His recognizes the peptide epitope, HHHHHH. This antibody was purified using protein-G chromatography and was subsequently immobilized onto a Sepharose™ Fast Flow matrix.Product Details
- 6xHis Tag
- Antibody Type
- Monoclonal 6-His antibody was raised against a recombinant protein containing the sequence HHHHHH.
- 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 6-His-tagged fusion proteins from crude starting material. On an analytical scale, it can also be used for immunoprecipitating His-tagged proteins.
Sepharose is a trademark of GE Life Sciences
- Application References
- Field J, et al. 1988. Mol Cell Biol. 8:2159.
AB_2564996 (BioLegend Cat. No. 900501)
- Biology Area
- Cell Biology
- Gene ID
- View information about 6-His on UniProt.org
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