SKU: CBS18
vTAG™ ANTI-HUMAN FLAG TAG ANTIBODY
NO WASH, QUANTITATIVE FLAG MEASUREMENT BY vFC™

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Conjugate(s): PE
Est. Limit of Detection: 30 Molecules per EV

 

Cargo analysis using vTag™ antibodies should be accompanied by a vesicle specific analysis method such as vFC™. vTag™ antibodies bundled with the vFC™ assay will be discounted 25%.

Certificate of Analysis

TDS MSDS

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Tested Reactivity: Tag sequence (DYKDDDDK).
Conjugate: PE (see dropdown at the top for available options).
Sensitivity: Limit of detection is estimated at 30 molecules per vesicle in vFC™ for the PE conjugate. Change selection above for LOD of the antibody conjugated to a different fluorophore. LOD is estimated using antibody binding beads and will vary depending upon sample, instrument, and other experimental conditions.
Application: Optimized for vFC™ with no wash steps required.
Clonality: Monoclonal.
Clone: L5.
Host and Isotype: Rat IgG2a.
Positive Control Sample: In progress.  We currently recommend testing antibody using Cellarcus’ antibody capture beads to ensure system is able to detect EV relevant numbers of anti-FLAG antibody.
Localization of Staining: Vesicle Surface.
Buffer and Stabilizer: 10mM PBS with 0.05% BSA & 0.09% azide.
Concentration: 5 µl/test.
Purification Method: Affinity purified during antibody production, additionally purified during conjugation to remove free dye and unbound antibody.
Immunogen: DYKDDDDK tagged recombinant mouse protein.
Storage Conditions: Store undiluted between 2°C and 8°C. Protected from prolonged exposure to light. Do not freeze. Stable for 1 year when properly stored.

FLAG Tag Immunostaining in vFC™?

This protocol provides a standardized means of analyzing size, concentration, and FLAG expression in EVs engineered to express FLAG. This protocol is built on vFC™ (#CBS4), an assay that enables specific detection and characterization of individual extracellular vesicles such as exosomes and microvesicles on commercially available flow cytometers. Because vFC™ is vesicle specific, we can characterize vesicles and cargo in a highly-reproducible, no-wash assay without artifacts from debris or reagent aggregates. In this protocol, vesicles will be stained using our vTag™ Anti-FLAG tag Antibody. See protocol 2 in the vFC™ kit handbook for additional considerations and full protocol details.

Procedure

  1. Dilute sample containing EVs to approximately 5×106 extracellular vesicles per µl. To determine vesicle concentration for an unknown sample, see supporting protocols included in the vFC™ kit.
  2. Prepare a sufficient amount of vFluor™ Red for all samples and controls (5 µl per well) according to kit instructions
  3. In duplicate, in the included 96 well plate, add 35 µl of dilution buffer, 5µl of prepared vFluor™ Red, 5 µl of vTag™ anti-FLAG tag antibody, and 5 µl of EV samples.
  4. Incubate in the dark at room temperature for 1 hr.
  5. Perform serial dilution of the samples as described in the assay handbook. Briefly, samples are to be diluted from between 1:100 to 1:1000 from staining concentration depending upon the flow cytometer being used.
  6. Run the diluted sample of labeled EVs on a qualified flow cytometer.

FLAG Tag is a peptide sequence that is used to tag recombinant proteins. The tag (sequence DYKDDDDK), is a non-natural antigen that is incorporated into proteins to simplify the labeling, detection, a protein purification via high affinity antibodies that have been developed against this sequence. It is preferable to other tags because the short sequence is not likely to affect the structure or function of the modified proteins.

In the context of engineered extracellular vesicles, incorporation of FLAG tags may be a viable option for quantitation of proteins of interest where there is no suitable antibody available.

vFC™ VESICLE FLOW CYTOMETRY EV ANALYSIS ASSAY
FOR COUNTING AND SIZING VESICLES IN MEDIA OR BUFFER

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Summary

Sample Format:Vesicles from cells in culture
Vesicle Size:>70nm
Certificate of Analysis

Protocol Handbook MSDS
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vCAL™ ANTIBODY CAPTURE NANOBEADS

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$350.00
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NO WASH, QUANTITATIVE FLAG MEASUREMENT BY vFC™