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HTRF® Anti-tag reagents toolbox

A wide range of anti-tag HTRF fluorophore conjugates for molecular interaction study

Tagged biomolecules are a key element in studying molecular interactions and offer researchers flexibility in assay design. The most frequently used tags for developing assays consist of proteinic or peptidic structures such as GST, 6HIS, c-myc, FLAG® and HA, maltose binding protein (MBP) or small organic motifs like dinitrophenyl (DNP). HTRF has also been successfully applied to NEB's SNAP-tag® technology.

The Lumi4®-Terbium cryptate conjugates bring an additional level of flexibility, as they can be combined either with the HTRF tracers, XL665 or d2, or with other fluorophores like fluorescein or green fluorescent protein (GFP). What's more, Lumi4-Terbium cryptate reagents enable KF free assays and are fully compatible with HTRF certified readers.

Features

  • High affinity monoclonal and polyclonal antibodies
  • Resistant to most buffer conditions and additives (e.g. DMSO, pH, chelators, ionic strength)
  • Compatible with membrane and cell-based assays
  • Lyophilized for easy handling and long term storage
  • Proven batch-to-batch consistency

Applications

  • Molecular interaction studies
  • Nuclear receptor screening
  • Investigation of enzymatic processes

Build HTRF assays for your specific application:

Reagents are sold by the number of tests (384-well, 20 µl reactions). Antibodies conjugated to XL665 or d2 are supplied on the basis of 20ng of antibody per well. The amount of active moiety per vial is also provided (as well as the number of tracers per vial - see product description sheet). The active moiety is defined as the active part of a conjugate (e.g. antibody).
 

How do the number of tests relate to active moiety?

The average conjugate quantity per well is information that reflects overall biological material content. For Eu3+ cryptate and biotin conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible.

For XL665 labeled entities, however, the amount of active moiety will not equal the amount of total conjugate. In practice, using the active moiety amount is generally preferred to the quantity of total conjugate as a basis for calculating reagent usage per well during assay development. The quantity of total conjugate supplied by Cisbio Bioassays will vary depending on the final molar ratio of the XL665 conjugate. The amount of active moiety, however, is constant and based on the number of tests ordered.

 

Custom labeling and assay development services are also available to help meet your research needs.

 

 

Motif/Species Antibody Active Moiety/5,000 tests Species and Subtype Specificity
GST MAb GSS11 10-15 µg Mouse IgG2a Schistosoma japonicum GST
6HIS MAb HIS-1 10-20 µg Mouse IgG2a HexaHistidine peptide
cMYC MAb 9E10 5-10 µg Mouse IgG1 EQKLISEEDL peptide
FLAG® MAb M2 5-10 µg Mouse IgG1 DYKDDDDK peptide
HA MAb HAS01 5-10 µg Mouse IgG1 YPYDVPDYA peptide
DNP MAb 265.5 ~ 20 µg Mouse IgG1 2.4-dinitrophenyl motif
MBP (Maltose Binding Protein) MAb MBP-17 5-10 µg Mouse IgG1 MBP or MBP fusion protien

 

The HTRF toolbox reagents include a large selection of anti-tag antibodies labeled with the HTRF tracers (Europium or Lumi4-Terbium Cryptates) and XL665 or d2 for detecting the most commonly used tags. These reagents enable assay development for a huge number of target classes and assay formats, such as protein: ligand or protein: protein interactions, nuclear receptor screening, receptor dimerization, kinase and protease assays and investigation of other enzymatic processes.

 

HTRF anti-GST for protein: protein interaction

 p53, a tumor suppressor protein activated in response to DNA damage, is regulated by the binding of HDM2 which induces ubiquitin-medicated degradation of p53. Our HTRF assay was developed to monitor p53/HDM2 binding, to assess the effect of serine phosphorylation within the p53 N-terminus on HDM2 binding, and to determine the relative affinity of a p53 homologue, p73, for HDM2. This assay employs a site-specific biotinylated p53 protein, a GST-fused HDM2 protein, streptavidin-XL665 and europium cryptate-labeled anti-GST antibody.

HTRF p53/HDM2 assay results are reported in Kane et al. - Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence. Anal Biochem. 2000; 278: 29-38.

 

Lumi4-Terbium for HTRF GFP and cmyc fused protein assay

Lumi4-Terbium Cryptate (Tb) conjugates are compatible with either HTRF acceptors such as XL665 or d2, or fluorescein and green fluorescent protein (GFP), meaning assay design flexibility can be considerably extended. As an example, HTRF anti-cmyc antibody labeled with Lumi4-Tb was used for the detection of GFP-and cmyc- fused peptide. A specific signal was only observed with cells expressing GFP and c-myc fused protein. Moreover, using Lumi4-Terbium Cryptate, the presence of KF in the detection buffer is not mandatory for the measurement. This is an advantage when ionic strength needs to be maintained at a low level for the interaction to occur. This combination opens new perspectives for the development of HTRF cell-based assays using encoded compatible fluorophores such as GFP.

HTRF GFP- and cmyc fused protein assay. Living cells were dispensed into a 384-well plate and transiently transfected with plasmid encoding for GFP-and cmyc fused peptide. After a 24h incubation at 37ºC, anti-cmyc-Lumi4-Tb in lysis buffer was added, and the signal was read on PHERAstar after a further hour of incubation at RT.  A signal to background higher than 3 was observed when comparing cells transfected with GFP-c-myc plasmid to the empty vector.

 

Ordering Info

DescriptionCat. noProduct insertMSDS
MAb Anti HA-d2 - 5,000 tests610HADAApdfpdf
MAb Anti HA-d2 - 20,000 tests610HADABpdfpdf
MAb Anti HA-K - 5,000 tests610HAKLApdfpdf
MAb Anti HA-K - 20,000 tests610HAKLBpdfpdf
MAb Anti HA-Tb - 5,000 tests610HATAApdfpdf
MAb Anti HA-Tb - 20,000 tests610HATABpdfpdf
MAb Anti HA-XL - 5,000 tests610HAXLApdfpdf
MAb Anti HA-XL - 20,000 tests610HAXLBpdfpdf
MAb Anti DNP-K - 5,000 tests61DNPKLApdfpdf
MAb Anti DNP-K - 20,000 tests61DNPKLBpdfpdf
MAb Anti DNP-XL665 - 5,000 tests61DNPXLApdfpdf
MAb Anti DNP-XL665 - 20,000 tests61DNPXLBpdfpdf
MAb Anti Flag M2-d2 - 5,000 tests61FG2DLA--
MAb Anti Flag M2-d2 - 20,000 tests61FG2DLB--
MAb Anti FLAG M2-K - 5,000 tests61FG2KLApdfpdf
MAb Anti FLAG M2-K - 20,000 tests61FG2KLBpdfpdf
MAb Anti FLAG M2-Tb - 5,000 tests61FG2TLApdfpdf
MAb Anti FLAG M2-Tb - 20,000 tests61FG2TLBpdfpdf
MAb Anti FLAG M2-XL665 - 5,000 tests61FG2XLApdfpdf
MAb Anti FLAG M2-XL665 - 20,000 tests61FG2XLBpdfpdf
MAb Anti GST-d2 - 5,000 tests61GSTDLApdfpdf
MAb Anti GST-d2 - 20,000 tests61GSTDLBpdfpdf
MAb Anti GST-K - 5,000 tests61GSTKLApdfpdf
MAb Anti GST-K - 20,000 tests61GSTKLBpdfpdf
MAb Anti GST-Tb - 5,000 tests61GSTTLApdfpdf
MAb Anti GST-Tb - 20,000 tests61GSTTLBpdfpdf
MAb Anti GST-XL665 - 5,000 tests61GSTXLApdfpdf
MAb Anti GST-XL665 - 20,000 tests61GSTXLBpdfpdf
MAb Anti 6HIS-d2 - 5,000 tests61HISDLApdfpdf
MAb Anti 6HIS-d2 - 20,000 tests61HISDLBpdfpdf
MAb Anti 6HIS-K - 5,000 tests61HISKLApdfpdf
MAb Anti 6HIS-K - 20,000 tests61HISKLBpdfpdf
MAb Anti 6HIS-Tb - 5,000 tests61HISTLApdfpdf
MAb Anti 6HIS-Tb - 20,000 tests61HISTLBpdfpdf
MAb Anti 6HIS-XL665 - 5,000 tests61HISXLApdfpdf
MAb Anti 6HIS-XL665 - 20,000 tests61HISXLBpdfpdf
Anti Kappa d2 - 5,000 tests61KAPDAA--
Anti Kappa d2 -20,000 tests61KAPDAB--
Anti Kappa K - 5,000 tests61KAPKAA--
Anti Kappa K -20,000 tests61KAPKAB--
MAb Anti MBP-d2 - 5,000 tests61MBPDAApdfpdf
MAb Anti MBP-d2 - 20,000 tests61MBPDABpdfpdf
MAb Anti MBP-K - 5,000 tests61MBPKAApdfpdf
MAb Anti MBP-K - 20,000 tests61MBPKABpdfpdf
MAb Anti MBP-Tb - 5,000 tests61MBPTAApdfpdf
MAb Anti MBP-Tb - 20,000 tests61MBPTABpdfpdf
MAb Anti cmyc-d2 frozen - 5,000 tests61MYCDAApdfpdf
MAb Anti myc-d2 frozen - 20,000 tests61MYCDABpdfpdf
MAb Anti cmyc-K - 5,000 tests61MYCKLApdfpdf
MAb Anti cmyc-K - 20,000 tests61MYCKLBpdfpdf
MAb Anti cmyc-Tb - 5,000 tests61MYCTAApdfpdf
MAb Anti cmyc-Tb - 20,000 tests61MYCTABpdfpdf
MAb Anti cmyc-XL665 - 5,000 tests61MYCXLApdfpdf
MAb Anti cmyc-XL665 - 20,000 tests61MYCXLBpdfpdf

Companion products

DescriptionCat. noProduct insertMSDS
Streptavidin-XL665 - 5,000 tests610SAXLApdfpdf
Streptavidin-XL665 - 20,000 tests610SAXLBpdfpdf
Streptavidin-XL665 - 3 mg610SAXLG-pdf
Streptavidin-XLent! - 5,000 tests611SAXLApdfpdf
Streptavidin-XLent! - 20,000 tests611SAXLBpdfpdf

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