フリーを含むユビキチン（モノ、ポリ）の定量

 

 

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The UbiQuant™ ELISA is a sandwich assay in which total ubiquitin is captured in the wells of a precoated microtiter plate using a proprietary ubiquitin binding reagent.  Unbound protein is removed by washing and then an anti-ubiquitin monoclonal antibody (VU-2) is added.  After removing unbound VU-2, bound antibody is measured using an enzyme linked anti-mouse antibody.

 

Intended Use

LifeSensors’ UbiQuant™ ELISA is intended for the accurate determination of the concentration of total ubiquitin (poly- + mono-) in biological samples including, cell lysates, tissue homogenates, and plasma. The kit is designed for research use only and is not intended forhuman or animal diagnostic or therapeutic applications.

 

Principle of the assay

The UbiQuant™ ELISA is a sandwich assay in which total ubiquitin is captured in the wells of a precoated microtiter plate using a proprietary ubiquitin binding reagent. Unbound protein is removed by washing and then an anti-ubiquitin monoclonal antibody (VU-2) is added. After removing unbound VU-2, bound antibody is measured using an enzyme linked anti-mouse antibody.

 

Components

Unless otherwise noted, all components should be stored at 4°- 8°C.

Coated plates: One precoated strip plate is provided. The plate is dried and vacuum packed. If not used within one month remove plate and store at -80°C.

Strip holder: One empty strip plate frame is provided.

Primary Antibody (VU-2): 1 vial of a 200x concentrate of mAb VU-2 in PBS containing 0.02% NaN3.

Standards (pUb): 2 vials of lyophilized ubiquitin standard (chains + free).The vial contains 5 μg of total ubiquitin.

Sensitizing Reagent 1 (SR1): 2 mL of a 20x stock (N.b. contains glutaraldehyde)

Sensitizing Reagent 2 (SR2): 4 mL of an 8x stock

Blocking concentrate (BC): 8 mL of 5X protein free blocking agent

Secondary antibody: 1 vial of 200X anti-mouse HRP conjugate

Plastic plate seals: Four provided

Detection reagent 1 (DR1): 0.5 mL vial of luminol reagent

Detection reagent 2 (DR2): 0.5 mL vial of stabilized peroxide reagent

DUB inhibitor (PR619): 1 vial containing 5 mg of a general deubiquitylase inhibitor (PR619; LifeSensors Cat. No. SI9619)

Standard curve

                       

 

Assay of biological samples

Limitations of Assay

Intraassay variability of the assay was tested by measuring twenty-one replicates of a 1 μg/mL pUb std. The concentration determined was 1.11 ± 0.1 μg/mL yielding a CV of 9.4%. These results are shown in the Table below.

Product Name	Product No.	Size
UbiQuant™ quantitative ubiquitin ELISA	UE101	1 (96 well)

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