iNOS Cell-Based ELISA Kit

iNOS Cell-Based ELISA Kit: 1) Cell Line: The cell line must express the target protein. This protocol can be used directly for adherent cells. For suspension cells and loosely attached cells, two steps are required: Coat the plates with 100 micro L of 10 micro g/mL Poly-L-Lysine to each well of the 96-well plate for 30 minutes at 37 degree C before proceeding to Step 1 of Assay Protocol. Use 8% formaldehyde to fix the cells on Step 5 of Assay Protocol.2) Cell Number and Sensitivity: The number of cells plated onto the 96-well plates depends on the expression level of INOS protein in the cells, cell size, treatment conditions and incubation time. The cells used for testing should be around 75-90% confluent. Typically for HeLa cells, seed 30,000 cells per well overnight for treatment the following day. The INOS Colorimetric Cell-Based ELISA Kit can detect INOS expression in as little as 5,000 HeLa cells.3) Cell Treatment: The cells can be treated with inhibitors, activators, stimulators (ie. chemicals, proteins/peptides) or a combination of the substances listed above. The cells can be treated with UV and serum starvation to meet the needs of the end-user.4) Positive and Negative Controls: Mouse Anti-GAPDH antibody (included) should be used to detect the internal positive controls for normalization of OD values of the target protein. The negative controls are HRP-Conjugated Anti-Rabbit IgG antibody and HRP- Conjugated Anti-Mouse IgG antibody alone in different wells (without the primary antibodies). Both positive and negative controls should be performed in the same plate with the INOS target experiments.5) Accuracy and Precision: Each condition should be performed in duplicate or in triplicate.

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