A simple assay for quantifying the expression level of proteins in situ is presented. The assay is performed directly in culture plates and simply requires incubating the cells (either fixed or in live cultures) with the appropriate antibody against the desired protein. Cells are then incubated with a peroxidase-conjugated secondary antibody followed by incubation with the soluble peroxidase substrate - tetramethylbenzidine. Antibody binding levels are quantified in an ELISA-based protocol by reading optical density at 450 nm. The assay is applicable for any surface-attached cell culture. The assay is highly sensitive, allowing detection of expressed proteins in a monolayer of cell cultured in a 96 well plate. The assay was optimized for a neuronal cell-line-P19. These are embryonal carcinoma cells which differentiate into neurons after treatment with retinoic acid. The neurons do not survive well in 96 well plates and are thus plated in 24 or 48 well plates. We demonstrate the use of the method for an in vivo labeling of neurons by quantifying epitopes which are exposed upon nerve stimulation. Advantages of a routine ELISA protocol in quantification over wide range of sensitivity and in performing simultaneous large number of repetitions and different controls are thus fully extended to live cell cultures.
Bibliographical noteFunding Information:
We thank Amichai Citri for an excellent assistance in optimization the in vivo assay in P19 neurons. We thank E. Heldman (Nes Ziona, Israel) for pharmacological analysis of CHO muscarinic receptor transfected cells and for mAChR antibodies. This work was supported by the ICRF and by the National Institute for Psychobiology in Israel and the Smith laboratory.
- Immunological assay