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Antibodies

AbFlex® AM-Tag antibody (rAb)

Catalog No: 91111 Format: 100 µg $375 Buy Now
Catalog No: 91112 Format: 10 µg $75 Buy Now

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Antibody Type:Recombinant
Isotype:IgG2a
Purification:Ni-NTA
Host:Mouse
Molecular Weight:15 kDa
Reactivity:Other (Wide Range)

Applications

ChIP Validated Western Blot Validated Enzyme-linked Immunosorbent Assay Validated

for AbFlex AM-Tag antibody (rAb)Application Notes


Validated Applications:
ChIP: 10 µg per ChIP
WB: 0.5 - 2 µg/ml dilution
Bead-based ELISA: 7 - 560 ng/ml

for AbFlex AM-Tag antibody (rAb)Immunogen

This antibody was raised against Active Motif's unique AM-Tag sequence for use with Tag-ChIP-IT® (Cat No. 53022) analysis.

for AbFlex AM-Tag antibody (rAb)Buffer

Purified IgG in 200 mM Hepes pH 7.5, 100 mm NaCl, 50 mM NaOAc, 30% glycerol and 0.035% sodium azide.

 
AbFlex<sup>®</sup> AM-Tag antibody (rAb) tested by ChIP.

AbFlex® AM-Tag antibody (rAb) tested by ChIP.
Active Motif’s pAM_1C_JunD Vector (Catalog No. 53044) was transiently transfected, mock transfected or transfected with pAM_1C Empty Vector (#53023) into HCT116 cells. Chromatin was harvested according to the instructions in the Tag-ChIP-IT® Kit (#53022). 10 µg of the AM-Tag antibody was used to immunoprecipitate the cross-linked AM-Tag-JunD fusion protein. qPCR data shows enrichment of AM-Tag-JunD with the FOXRED qPCR primer set and little to no enrichment in the mock transfections, empty vector transfections, or when using the negative control Unt12 qPCR primer set.

AbFlex<sup>®</sup> AM-Tag antibody (rAb) tested by Western blot.

AbFlex® AM-Tag antibody (rAb) tested by Western Blot.
Active Motif’s pAM_1C_JunD Vector (Catalog No. 53044) was transfected, mock transfected or transfected with pAM_1C Empty Vector (Catalog No. 53023) into HeLa or HCT116 cells using 10 µg DNA and 30 µl FuGENE transfection reagent, 48 hours post-transfection nuclear lysates were prepared. 20 µg lysate was loaded per well. Western blot was performed using a 1:500 dilution of AM-Tag antibody.

for AbFlex AM-Tag antibody (rAb)Background

AbFlex® antibodies are recombinant antibodies (rAbs) that have been generated using defined DNA sequences to produce highly specific, reproducible antibodies. Each AbFlex antibody contains a 6xHis-Tag, an avidin tag sequence for enzymatic biotin conjugation using the biotin ligase, BirA, and a sortase recognition motif (LPXTG) to attach a variety of labels directly to the antibody including fluorophores, enzymatic substrates (HRP, AP), peptides, drugs as well as solid supports.

AbFlex® AM-Tag antibody was expressed as full-length IgG with mouse immunoglobulin heavy and light chains (IgG2a isotype) in mammalian 293 cells.

The AM-Tag antibody is specifically designed for use with Active Motif’s unique AM-Tag sequence in combination with the Tag-ChIP-IT® Kit (Catalog No. 53022). Simply clone your protein of interest into Active Motif’s pAM_1C Empty Vector (Catalog No. 53023), or add the AM-Tag sequence to the C-terminus of your protein expression vector. Following transfection, cell lysates are prepared and screened by Western blot for expression of the AM-Tag fusion protein using the AM-Tag antibody. Optimization may be required to identify the optimal conditions for transfection and protein expression.

Once transfection conditions have been optimized, the Tag-ChIP-IT® Kit can be used to isolate chromatin and perform ChIP. The AM-Tag has minimal cross reactivity with mammalian samples to ensure low background signal. The tag is also unstructured, which allows the tag to protrude from the protein of interest for maximum exposure during immunoprecipitation. This increases the enrichment efficiency of low abundance transcription factors for more reliable and consistent ChIP results.

Use of the AM-Tag for chromatin immunoprecipitation avoids the need for protein-specific ChIP-validated antibodies. It also enables the analysis of sequence variants, mutations and truncations on gene regulation.

Chromatin IP Assays

This antibody has been validated for use in ChIP and/or ChIP-Seq, and can be used with Active Motif's ChIP-IT® High Sensitivity Kit or our magnetic bead-based ChIP-IT® Express Kits. For an overview of all of our ChIP products, please visit our Chromatin IP Page to learn about ChIP products designed for use with AbFlex® AM-Tag antibody (rAb).

Bridging Antibody to Improve ChIP, MeDIP and IP

Some isotypes of mouse IgG antibodies do not bind well to protein G-conjugated agarose/magnetic beads, which are commonly used in ChIP, MeDIP and IP experiments. Capture efficiency can be improved by including an anti-mouse IgG bridging antibody because it binds with a strong affinity to both the protein G beads and the mouse IgG antibody. By increasing the affinity of the mouse primary antibody for the protein G bead, the Bridging Antibody for Mouse IgG can help improve the results of all ChIP, MeDIP and IP experiments that use AbFlex® AM-Tag antibody (rAb).

for AbFlex AM-Tag antibody (rAb)Storage

Some products may be shipped at room temperature. This will not affect their stability or performance. Upon receipt, unconjugated antibodies may be stored at -20°C for up to 2 years. Fluorophore- & enzyme-conjugated antibodies should be stored at 4°C. Fluorophore-conjugated antibodies should be protected from light. Keep reagents on ice when not in storage; to avoid repeated freeze/thaw cycles, we recommend aliquoting items that will be stored frozen into single-use fractions prior to freezing.

for AbFlex AM-Tag antibody (rAb)Guarantee

This product is guaranteed for 6 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.

Application Key

  • ChIP = Chromatin Immunoprecipitation;
  • DB = Dot Blot;
  • ELISA = Enzyme-linked Immunosorbent Assay;
  • EMSA = Electrophoretic Mobility Shift Assay
  • FC = Flow Cytometry;
  • ICC = Immunocytochemistry;
  • IF = Immunofluorescence;
  • IHC = Immunohistochemistry;
  • IP = Immunoprecipitation;
  • MeDIP = Methyl-DNA Immunoprecipitation;
  • TR-FRET = Time-Resolved Fluorescence Resonance Energy Transfer;
  • WB = Western Blotting
 

Technical Data Sheet

AbFlex® AM-Tag antibody (rAb)
 

Data Thumbnails

AbFlex<sup>®</sup> AM-Tag antibody (rAb) tested by ChIP.

ChIP of AbFlex® AM-Tag antibody (rAb).
(Click image to enlarge and see details.)

AbFlex<sup>®</sup> AM-Tag antibody (rAb) tested by Western blot.

Western blot of AbFlex® AM-Tag antibody (rAb).
(Click image to enlarge and see details.)