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
