This ELISA kit has not been validated for lysates, but it is expected to work. Follow the instructions below for preparing samples.
Cell Lysates:
Rinse cells with PBS, ensuring all PBS is removed before detaching cells. Detach 2 x 10^7 cells by trypsinization or scraping and transfer to a microfuge tube. Centrifuge to pellet the cells and remove any remaining buffer. Add approximately 500 µl of prepared lysis buffer and pipette up and down to resuspend the pellet. Incubate the lysates with shaking at 4°C for 30 minutes. Spin down the tubes in a microfuge at top speed (10,000g) for 10 minutes at 4°C, and transfer the supernatants to a clean tube.
For lysis buffer, use 43-040 Cell Lysis buffer or 20-188 RIPA lysis buffer.
Guidelines for lysis buffer composition:
Avoid using more than 0.1% SDS or other strongly denaturing detergents. Non-ionic detergents such as Triton X-100 or NP-40 are recommended, although zwitterionic detergents like CHAPS, or mild ionic detergents such as sodium deoxycholate, will work.
Use no more than 2% v/v total detergent.
Avoid sodium azide.
Avoid using more than 10 mM reducing agents, such as dithiothreitol or mercaptoethanols.
Adding a protease inhibitor cocktail to the lysis buffer prior to homogenization is strongly recommended. Inhibitor cocktails like P2714 and P8465 can be purchased and used according to their specifications.
Lysate Application:
Use cell or tissue lysates immediately or aliquot and store at –80°C. Avoid repeated freeze-thaw cycles. Keep thawed lysates on ice prior to use. For the first experiment, perform serial dilution testing, starting with a 5-fold dilution, to determine the optimal protein load for the assay. Optimal dilution depends on the abundance of target proteins and should be determined empirically. Determine the total protein concentration using the Pierce BCA Protein Assay Kit, Cat#: 23227. Dilute lysate to a final total protein concentration of 50-500 µg/ml (5-fold or more is preferred) when performing antibody array or ELISA testing.
Dilute the cell lysate for this kit with Assay Diluent B. For other ELISA kits validated with cell lysate samples, a minimum 5-fold dilution is recommended to avoid sample matrix effects, but the optimal sample dilution must be determined empirically by the researcher.
