Anti-6-His Epitope Tag Affinity Matrix (Previously Covance catalog# AFC-156P)

Product Details

Verified Reactivity

Epitope tag

Reported Reactivity

Species independent

Antibody Type

Monoclonal

Host Species

Mouse

Immunogen

Monoclonal 6-His antibody was raised against a recombinant protein containing the sequence HHHHHH.

Formulation

Purified IgG immobilized on Sepharose™ Fast Flow beads (in PBS + 0.03% Thimerosal).

Preparation

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.

Application

Purification, IP

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

(PubMed link indicates BioLegend citation)

1. Field J, et al. 1988. Mol Cell Biol. 8:2159.

RRID

AB_2564996 (BioLegend Cat. No. 900501)

Antigen Details

Biology Area

Cell Biology

Gene ID

NA

UniProt

View information about 6-His on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Other Formats

Certificate of Analysis