HTRF MAb Anti FLAG M2 XL665-WEICHILAB

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.

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Assay principle

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.

Assay protocol

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.

Assay details

Build HTRF interaction assays for your specific application

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).

How do the number of tests relate to active moiety?

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

Recommended quantities of Cryptate and XL665 conjugates

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.

At Revvity, we regard "impossibility" as inspiration and "inability" as the driving force. Revvity offers health science solutions, cutting - edge technologies and professional services, with its business covering the entire end - to - end process of scientific research exploration, development, diagnosis, and treatment. Relying on years of in - depth cultivation in fields such as translational multi - omics technology, biomarker identification, imaging, disease prediction, screening, detection and diagnosis, and informatics, Revvity is using the power of technology to break through the boundaries of human potential.

In 2023, Revvity's turnover exceeded $2.7 billion. It has more than 11,000 employees worldwide and provides diverse services for customers such as pharmaceutical and biotechnology enterprises, diagnostic laboratories, scientific research institutions and government agencies. The company is a member of the S&P 500 Index, and its customers are spread across more than 190 countries and regions around the world.

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Cat.NO	Name	Size
61FG2XLB	HTRF MAB ANTI FLAG M2-XL665	20K PTS

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Specifications

Assay principle

Assay protocol

Assay details

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