2X Lysis Buffer Preparation Instructions
2X Lysis Buffer should be diluted 2-fold with deionized or distilled water. Supplementation with protease or phosphatase inhibitors is recommended. You may use RayBiotech Protease Inhibitor Cocktail (AA-PI
), RayBiotech Phosphatase Inhibitor Cocktail Set I (AA-PHI-I
) or Phosphatase Inhibitor Cocktail Set II (AA-PHI-II
), or the RayBiotech Phosphatase / Protease Inhibitor Set (AA-PIPHI
). Alternatively, you can purchase inhibitor cocktails from another manufacturer and follow their specifications.
Rinse cells with PBS, taking care to remove any remaining PBS before detaching cells. Detach 2 x 107 cells by trypsinization or scraping and transfer the cells to a microfuge tube. Centrifuge to pellet the cells and then remove any remaining buffer. Add ~500 µl 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 for 10 minutes at (4°C), and transfer the supernatants to a clean tube.
Transfer approximately 100 mg crude tissue into a tube with 1 ml 1X Lysis Buffer. Homogenize the tissue according to homogenizer manufacturer instructions. Transfer extracts to microfuge tubes and centrifuge at top speed for 20 minutes (4°C).
Cell or tissue lysates should be used immediately or aliquoted and stored at –80°C. Avoid repeated freeze-thaw cycles. Thawed lysates should be kept on ice prior to use.
For the first experiment, we recommend 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.
The total protein concentration may be determined using the BCA Protein Assay Kit from Pierce, Cat#: 23227. We recommended to dilute your lysate to a final total protein concentration of 50-500 ug/ml (5-fold or more is preferred) when performing antibody array or ELISA testing.
Store at 4°C for 6 months or frozen at -80°C for 1 year