Original
Cell-Based Infrared ELISAs: Intracellular Protein Detection Without Lysate Preparation
DOI: 10.2144/000113790

Abstract




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R&D Systems Cell-Based Infrared ELISAs simultaneously measure the levels of two proteins in the same microplate well in whole cells, eliminating the need for lysate preparation. Kits utilize LI-COR® IRDye® 800CW- and IRDye® 680LT-conjugated secondary antibodiesa

aThis product is covered by US 6,995,274; US 7,504,089; foreign equivalents and patents pendingto detect the protein of interest and a second housekeeping protein in adherent or non-adherent cells, allowing for normalization of the target protein in each well. Data are presented that show detection of cleaved caspase-3 in staurosporine-treated Jurkat cells using the R&D Systems Cell-Based Infrared ELISA.


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Materials

Human Cleaved Caspase-3 (Asp175) Cell-Based Infrared ELISA (R&D Systems, Catalog #KCBIR835)

37% formaldehyde .(Molecular Biology Grade; Sigma, Catalog # F8775)

PBS .(Irvine Scientific, Catalog #9240)

Deionized or distilled water

Pipette tips and pipettes

Multi-channel pipette

37°C cell culture incubator

Microfuge tubes

Centrifuge with microtiter plate carriers

LI-COR Odyssey.®Infrared Imaging System, or equivalent

Methods

1. Culture, Stimulate, Fix, and Block Cellsb

bProtocol is for adherent cells. Please refer to package insert for procedure for non-adherent cells

■ Seed cells (10,000 – 20,000 cells) into each well of the 96-well microplate (provided in kit). Incubate overnight at 37°C.

■ Grow and treat cells per the experi-mental protocol.

■ Centrifuge the plate at 500 × g for 3 minutes at 2–8°C. Remove medium. Incubate cells in 4% formaldehyde in PBS for 20 minutes at room temperature.

■ Remove formaldehyde. Wash cells 3 times with 1 × Wash Buffer (provided in kit).

■ Incubate cells in Blocking Buffer (provided in kit) for 1 hour at room temperature.


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2. Binding of Primary Antibodies

■ Remove Blocking Buffer. Wash cells 3 times with 1 × Wash Buffer.

■ Incubate cells in the Primary Antibody Mixture (provided in kit) for 16 hours at 2-8°C.

3. Binding of Secondary Antibodies

■ Remove Primary Antibody Mixture. Wash cells 3 times with 1 × Wash Buffer.

■ Incubate cells in the Secondary.Antibody Mixture (provided in kit) for 2 hours at room temper- ature.

4. Near-Infrared Detection

■ Remove Secondary Antibody Mixture. Wash cells 4 times with 1 × Wash Buffer.

■ Image the plate by scanning simul - taneously at 700 nm and 800 nm with the LI-COR Odyssey Infrared Imaging System, or equivalent.

Results

The Jurkat human acute T cell leukemia cell line was treated with increasing concentrations of staurosporine. Cleaved caspase-3 (Asp175) and GADPH protein levels were determined using the Human Cleaved Caspase-3 (Asp175) Cell-Based Infrared ELISA (Catalog # KCBIR835)..

Contact

R&D Systems, Inc.
Telephone: 800-343-7475
E-mail:[email protected]

R&D Systems Europe Ltd.
Telephone: +44 (0) 1235-529449
E-mail:[email protected]

R&D Systems China Co., Ltd.
Telephone: (21) 52380373
E-mail:[email protected]



LI-COR, Odyssey, and IRDye are registered trademarks of LI-COR, Inc.

For research use only. Not for use in diagnostic procedures.

aThis product is covered by US 6,995,274; US 7,504,089; foreign equivalents and patents pending

bProtocol is for adherent cells. Please refer to package insert for procedure for non-adherent cells

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