vTAG™ ANTI-HUMAN CD235AB ANTIBODY
NO WASH, QUANTITATIVE CD235AB MEASUREMENT BY vFC™

$105.00$275.00

152 in stock

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
Clear
SKU: CBS31 Category:

Description

Tested Reactivity: Routinely tested against human CD235ab on Red Blood Cell-Derived EVs (CBS3).
Additional Reactivity: This clone also reacts with CD235ab from non-human primates including african green, and baboons and may be suitable for labeling vesicles from these species.
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.  LOD is estimated using antibody binding nanobeads and will vary depending upon sample, instrument, and other experimental conditions.
Application: Optimized for vFC™ with no wash steps required.
Clonality: Monoclonal.
Clone: HIR2.
Host and Isotype: Mouse IgG2b, κ.
Positive Control Sample: EVs from Human Red Blood Cells (RBCs).
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.
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.

CD235ab Immunostaining in vFC™

This protocol is a summary of steps required to quantify CD235ab on EVs via vFC™. A full version of this protocol is described in protocol 2 in the vFC™ (#CBS4) handbook and should be used for actual lab work as it contains necessary instrument calibration steps, data analysis protocols, and other information beyond the scope of this overview. vFC™ is 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-CD235ab antibody.

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-CD235 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.

CD235ab (CD235) is a pair of sialoglycoproteins also known as Glycophorin A and Glycophorin B. Glycophorin A is highly expressed on the membrane of red blood cells and is highly homologous to Glycophorin B. CD235ab is shed on EVs from red blood cells. Analysis of  red blood cells and platelets by vFC™ reveals baseline resolution of EVs from RBCs from control and no detectable staining on EVs from platelets, demonstrating specificity of the vTag™ antibody when paired with an appropriate assay.

Additional information

Size

20 Tests, 100 Tests

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NO WASH, QUANTITATIVE CD235AB MEASUREMENT BY vFC™

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