XL665-labeled anti-FLAG M2 antibody for capturing FLAG M2-tagged proteins in protein/protein interaction assays.
Important notice: Product update
Please be informed that references 61FG2XLF/A/B will be replaced by 61FGBXLF/A/B in the upcoming months. We recommend transitioning to the new products at your earliest convenience.
For research use only. Not for use in diagnostic procedures.
MAb Anti FLAG M2-XL665 is an IgG1 raised against FLAG® fusion proteins labeled with XL665. Unlike anti-FLAG®M1 antibody, the M2 antibody will recognize the FLAG® sequence at the N-terminus or C-terminus of FLAG® fusion proteins.
This reagent can be used in both biochemical and cellular formats to study a wide variety of interactions: protein/protein, protein/peptide, protein/DNA, protein/RNA, protein/carbohydrate, protein/small molecule, receptor/ligand.
HTRF can detect a broad range of affinity constants ranging from picomolar to low millimolar.
Application |
Protein-Protein Interaction
|
---|---|
Assay Target Class |
Binding Assay
|
Assay Technology |
TR-FRET
|
Brand |
HTRF
|
Detection Method |
HTRF
|
Product Group |
Fluorescent Reagent
|
Shipping Conditions |
Shipped Ambient
|
Therapeutic Area |
Cardiovascular
Infectious Diseases
Inflammation
Metabolism/Diabetes
NASH/Fibrosis
Neuroscience
Oncology & Inflammation
Rare Diseases
|
Unit Size |
1,000 Assay Points
|
In an HTRF interaction assay, one partner is labeled (directly or indirectly) with the donor, and the other with the acceptor (again, directly or indirectly). The intensity of the signal is proportional to the binding of the 2 partners. In the example shown here: MAb Anti FLAG M2-XL665 binds to the FLAG M2 tagged partner A while partner B* binds to a specific Ab labeled with an HTRF donor. *partner B can also be biotinylated, tagged, Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with donor for the detection.
The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a FLAG M2-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add MAb Anti FLAG M2-XL665 (5 µL) and anti-partner B labeled with donor (5 µL), incubate and read. *partner B can also be biotinylated, tagged, Fc fused or directly labeled. In these cases, use the corresponding HTRF reagent (anti-Tag, anti species, protA, Streptavidin) labeled with donor for the detection.
Reagents are sold by the number of tests (20 µL reactions). Antibodies conjugated to XL665 or d2 are supplied on the basis of 20 ng 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).
The average conjugate quantity per well reflects overall biological material content. Using the active moiety amount is generally preferred to the quantity of total conjugate. For Cryptate and d2 conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible. This is not the case for XL665 labeled entities for which the quantity of total conjugate will vary depending on the final molar ratio of the XL665 conjugate, however, the amount of active moiety, provided by Revvity, is constant and based on the number of tests ordered.
Motif/Species Antibody Active Moiety/5,000 tests Species and Subtype Specificity FLAG® MAb M2 5-10 µg Mouse IgG1 DYKDDDDK peptide
Cryptate conjugates must not be excessive in order to prevent reader saturation and an unacceptable level of background. In most cases, a cryptate concentration of 1 to 5nM is appropriate, and will generate 20,000 to 80,000 cps at 620 nm depending on the HTRF compatible reader used. The XL665 conjugate must match its assay counterpart as closely as possible in order for the maximum number of biomolecules to be tagged with the XL665 acceptor. Thus, to detect a tagged molecule at an assay concentration of 20nM, the concentration of anti-Tag-XL665 should be equimolar or higher.
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