HTRF® Anti-tag reagents

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.

Discover our latest Anti-6His Gold conjugates available with Tb and Europium tracers – Providing better sensitivity and assay windows for your molecular interaction studies.

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Product Specifications

Build HTRF Protein-Protein interaction (PPI) 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 IgG2b 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

Assay Performances

The HTRF PPI 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.

Product Applications

Optimal placement of the tag is protein-specific, and depending on this location the assay performance may vary from one protein to another. Here we describe the parallel use of different Cisbio anti 6His conjugates (Eu, Tb , Eu Gold, Tb Gold) and Lanthascreen anti His tag antibody (Eu, Tb). These cases reveal that depending on the interaction studied, the antibody used might need to be different: in one case the Eu based antibody performs well and in the other, terbium showed better results. For each interaction tested below, the Cisbio Anti6His Gold conjugate out-performed the Lanthascreen Anti-6His tag antibody.

1. Europium based antibodies to qualify BRD4(1)-H4 peptide interaction

Assay Principle

6His-tagged BRD4(1) reader domain binding to the biotinylated peptide substrate H4 is detected by a conjugate mix: donor Cryptate labeled antibody raised against the 6His tag and SA-XL665, for the detection of the biotin-peptide. (For both Cisbio and Lanthascreen antibodies, the assay windows were lower when using Terbium donor in this case (data not shown))

Step 1: Determination of optimal peptide concentration for further screening

The first step of peptide titration revealed that no detection was possible when using Lanthascreen Eu or Tb antibody donor s (data not shown for Tb labeling). For these, no further compound screening can be considered. However, both Gold and the classical version of anti-6His antibodies from Cisbio enabled detection of the interaction. The assay window was increased using the Gold version.

Step 2: BRD4(1)-H4 peptide interaction inhibitors screening : JQ1 dose-response

JQ1 inhibitor IC50 was found between 12 and 24.5nM ,in agreement with the literature. As expected, the new Anti-6His cryptate Gold showed an increased assay window .

2. Terbium based antibodies to qualify NEFcore/ Hck(SH3 domain) protein-protein interaction

Assay Principle

NEFcore – HcK SH3 domain interaction is an important target for HIV infectious disease. Inhibitors of this interaction are important targets and are considered as therapeutic solutions. NEFcore , fused to the GST tag is detected using Cisbio PPI MAb Anti GST-d2 antibody at 3 nM. The HcK SH3 domain which contains a 6His tag is detected using the Anti 6His Gold donor conjugate.

Step 1: Determination of optimal GST-Nef concentration for further screening

Where no detection was possible when using Cisbio Anti-6His Tb Cryptate (same results observed with Eu, data not shown), the Gold version showed a significant increase in the assay window, enabling further screening of this protein-protein interaction. The results were superior to those obtained using Lanthascreen Tb anti-HIS-tag antibody (same conclusion for Eu, data not shown).

Step 2: Protein protein interaction Inhibitor screening

Inhibitor screening assay is shown using FynSH3 protein, which binds to the Nef Core domain competing with the HcK SH3 domain. This showed an IC50 of 0.7-2 µM. The assay window was slightly better using Cisbio Anti-6His Tb cryptate.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
MAb Anti HA-d2 - 5,000 tests610HADAA
MAb Anti HA-d2 - 20,000 tests610HADAB
MAb Anti HA-d2 - 1,000 tests610HADAF
MAb Anti HA-Eu cryptate - 5,000 tests610HAKLA
MAb Anti HA-Eu cryptate - 20,000 tests610HAKLB
MAb Anti HA-Tb cryptate - 5,000 tests610HATAA
MAb Anti HA-Tb cryptate - 20,000 tests610HATAB
MAb Anti HA-Tb cryptate - 1,000 tests610HATAF
MAb Anti HA-XL665 - 5,000 tests610HAXLA
MAb Anti HA-XL665 - 20,000 tests610HAXLB
MAb Anti HA-XL665 - 1,000 tests610HAXLF
MAb Anti DNP-Eu cryptate - 5,000 tests61DNPKLA
MAb Anti DNP-Eu cryptate - 20,000 tests61DNPKLB
MAb Anti DNP-XL665 - 5,000 tests61DNPXLA
MAb Anti DNP-XL665 - 20,000 tests61DNPXLB
MAb Anti FLAG M2-d2 - 5,000 tests61FG2DLA
MAb Anti FLAG M2-d2 - 20,000 tests61FG2DLB
MAb Anti FLAG M2-d2 - 1,000 tests61FG2DLF
MAb Anti FLAG M2-Eu cryptate - 5,000 tests61FG2KLA
MAb Anti FLAG M2-Eu cryptate - 20,000 tests61FG2KLB
MAb Anti FLAG M2-Tb cryptate - 5,000 tests61FG2TLA
MAb Anti FLAG M2-Tb cryptate - 20,000 tests61FG2TLB
MAb Anti FLAG M2-Tb cryptate - 1,000 tests61FG2TLF
MAb Anti FLAG M2-XL665 - 5,000 tests61FG2XLA
MAb Anti FLAG M2-XL665 - 20,000 tests61FG2XLB
MAb Anti FLAG M2-XL665 - 1,000 tests61FG2XLF
MAb Anti GST-d2 - 5,000 tests61GSTDLA
MAb Anti GST-d2 - 20,000 tests61GSTDLB
MAb Anti GST-d2 - 1,000 tests61GSTDLF
MAb Anti GST-Eu cryptate - 5,000 tests61GSTKLA
MAb Anti GST-Eu cryptate - 20,000 tests61GSTKLB
MAb Anti GST-Tb cryptate - 5,000 tests61GSTTLA
MAb Anti GST-Tb cryptate - 20,000 tests61GSTTLB
MAb Anti GST-Tb cryptate - 1,000 tests61GSTTLF
MAb Anti GST-XL665 - 5,000 tests61GSTXLA
MAb Anti GST-XL665 - 20,000 tests61GSTXLB
MAb Anti GST-XL665 - 1,000 tests61GSTXLF
MAb Anti-6HIS-Eu cryptate Gold - 5,000 tests61HI2KLA
MAb Anti-6HIS-Eu cryptate Gold - 20,000 tests61HI2KLB
MAb Anti-6HIS-Tb cryptate Gold - 5,000 tests61HI2TLA
MAb Anti-6HIS-Tb cryptate Gold - 20,000 tests61HI2TLB
MAb Anti 6HIS-Tb cryptate Gold - 1,000 tests61HI2TLF
MAb Anti 6HIS-d2 - 5,000 tests61HISDLA
MAb Anti 6HIS-d2 - 20,000 tests61HISDLB
MAb Anti 6HIS-d2 - 1,000 tests61HISDLF
MAb Anti 6HIS-Eu cryptate - 5,000 tests61HISKLA
MAb Anti 6HIS-Eu cryptate - 20,000 tests61HISKLB
MAb Anti 6HIS-Tb cryptate - 5,000 tests61HISTLA
MAb Anti 6HIS-Tb cryptate - 20,000 tests61HISTLB
MAb Anti 6HIS-Tb cryptate - 1,000 tests61HISTLF
MAb Anti 6HIS-XL665 - 5,000 tests61HISXLA
MAb Anti 6HIS-XL665 - 20,000 tests61HISXLB
MAb Anti 6HIS-XL665 - 1,000 tests61HISXLF
MAb Anti MBP-d2 - 5,000 tests61MBPDAA
MAb Anti MBP-d2 - 20,000 tests61MBPDAB
MAb Anti MBP-Eu cryptate - 5,000 tests61MBPKAA
MAb Anti MBP-Eu cryptate - 20,000 tests61MBPKAB
MAb Anti MBP-Tb cryptate - 5,000 tests61MBPTAA
MAb Anti MBP-Tb cryptate - 20,000 tests61MBPTAB
MAb Anti cmyc-d2 - 5,000 tests61MYCDAA
MAb Anti cmyc-d2 - 20,000 tests61MYCDAB
MAb Anti cmyc-d2 - 1,000 tests61MYCDAF
MAb Anti cmyc-Eu cryptate - 5,000 tests61MYCKLA
MAb Anti cmyc-Eu cryptate - 20,000 tests61MYCKLB
MAb Anti cmyc-Tb cryptate - 5,000 tests61MYCTAA
MAb Anti cmyc-Tb cryptate - 20,000 tests61MYCTAB
MAb Anti cmyc-Tb cryptate - 1,000 tests61MYCTAF
MAb Anti cmyc-XL665 - 5,000 tests61MYCXLA
MAb Anti cmyc-XL665 - 20,000 tests61MYCXLB
MAb Anti cmyc-XL665 - 1,000 tests61MYCXLF

Companion products

DescriptionCat. noProduct insertMSDS
Streptavidin-XL665 - 5,000 tests610SAXLA
Streptavidin-XL665 - 20,000 tests610SAXLB
Streptavidin-XLent! - 5,000 tests611SAXLA
Streptavidin-XLent! - 20,000 tests611SAXLB
PPI - Terbium detection buffer - 200 mL61DB10RDF
PPI - Europium detection buffer - 200 mL61DB9RDF
6His Check kit Gold - 10,000 tests64HISPEH