Anti-Glu-Glu Epitope Tag Affinity Matrix (Previously Covance catalog# AFC-115P)

Pricing & Availability
Clone
Glu-Glu (See other available formats)
Other Names
AFC-115P-1000
Previously
Covance Catalog# AFC-115P
Isotype
Mouse IgG1
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Product Citations
publications
Glu-Glu_Glu-Glu_Epitope_Tag_Ascites_WB_060118
Immunoprecipitation of 293E cells transfected with or without multi-tag plasmid (The plasmid includes Glu-Glu, GST, HA, etc.). 800 µg cell lysates were immunoprecipitated with 30 µl matrix (Clone Glu-Glu). 1% (5 µg) cell lysates was tested as input. Western blot analysis was performed using Direct-Blot™ HRP anti-HA Antibody (Cat. No. 901519).
  • Glu-Glu_Glu-Glu_Epitope_Tag_Ascites_WB_060118
    Immunoprecipitation of 293E cells transfected with or without multi-tag plasmid (The plasmid includes Glu-Glu, GST, HA, etc.). 800 µg cell lysates were immunoprecipitated with 30 µl matrix (Clone Glu-Glu). 1% (5 µg) cell lysates was tested as input. Western blot analysis was performed using Direct-Blot™ HRP anti-HA Antibody (Cat. No. 901519).
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900301 1 ml $540
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Description

It is extremely specific and recognizes either six amino acid sequence, EYMPME, or EFMPME. This antibody was purified using protein-G chromatography and was subsequently immobilized onto a Sepharose™ Fast Flow matrix.

Product Details
Technical data sheet

Product Details

Reactivity
EYMPME and EFMPME Tags
Antibody Type
Monoclonal
Immunogen
The Glu-Glu monoclonal antibody was raised against the peptide sequence CEEEEYMPME derived from the polyoma virus medium T antigen
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. For immunoprecipitation, the suggested use of this reagent is 20 - 60 µl slurry per sample.

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 Glu-Glu epitope tagged fusion proteins from crude starting material. On an analytical scale, it can also be used for immunoprecipitating Glu-Glu epitope 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.
  2. McKay MM, et al. 2010. Methods Mol Biol. 661:323.
  3. Zheng X, et al. 2010. Genes Dev. 24:57. (IP) PubMed
  4. Cowling VH, et al. 2014. Oncogene. 33:3519.
Product Citations
  1. Zheng X, et al. 2010. Genes Dev. 24:57-71. PubMed
Publication Library
RRID
AB_2564995 (BioLegend Cat. No. 900301)

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Go To Top Version: 6    Revision Date: 06/29/2018

For research use only. Not for diagnostic use. Not for resale. BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products.

 

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This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
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