Human Total Bcl-2 DuoSet IC ELISA Summary

Product Summary

This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure total Bcl-2 in cell lysates. An immobilized capture antibody specific for Bcl-2 binds both phosphorylated and unphosphorylated Bcl-2. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.

Preparation and Storage

Background:  Bcl-2

Bcl-2 is a member of a family of proteins that regulates outer mitochondrial membrane permeability. Bcl-2 is an anti-apoptotic member that prevents release of cytochrome c from the mitochondria intermembrane space into the cytosol. Bcl-2 is present on the outer mitochondrial membrane and is also found on other membranes in some cell types. Natural Bcl-2 contains a carboxyl-terminal mitochondria targeting sequence.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.