Size: 5 ml

2X lysis buffer for ELISA kits

2X Lysis Buffer Preparation Instructions

The 2X Lysis Buffer should be diluted 2 times with deionized or distilled water. Supplementation with protease or phosphatase inhibitors is recommended. You can use RayBiotech Protease Inhibitor Cocktail (AA-PI), RayBiotech Phosphatase Inhibitor Cocktail I (AA-PHI-I), or Phosphatase II Inhibitor Cocktail (AA-PHI-II), or Phosphatase Inhibitor / RayBiotech protease (AA -PIPHI). Alternatively, you can buy inhibitor cocktails from another manufacturer and follow their specifications.

Preparation of the lysate

Cell lysates

Rinse cells with PBS, taking care to remove any remaining PBS before separating cells. Separate 2×107 cells by trypsinization or scraping and transfer cells to a microcentrifuge tube. Centrifuge to pellet the cells and then remove any remaining buffer. Add ~ 500 µl of prepared Lysis Buffer and pipet up and down to resuspend the pellet. Incubate the lysates with shaking at 4 ° C for 30 minutes. Spin the tubes in a microcentrifuge at maximum speed for 10 minutes at (4 ° C) and transfer the supernatants to a clean tube.

Tissue lysates

Transfer approximately 100 mg of crude tissue to a tube with 1 ml of 1X lysis buffer. Homogenize the tissue according to the homogenizer manufacturer’s instructions. Transfer the extracts to microcentrifuge tubes and centrifuge at maximum speed for 20 minutes (4 ° C).

Lysate application

• 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 before use.
• For the first experiment, we recommend serial dilution tests, starting with a 5-fold dilution, to determine the optimal protein load for the assay. The optimal dilution depends on the abundance of target proteins and must be determined empirically.
• Total protein concentration can be determined using Pierce’s BCA Protein Assay Kit, Cat #: 23227. We recommend diluting your lysate to a final total protein concentration of 50-500 ug/ml (5 times or more preferred) when performing ELISA or antibody matrix tests.

Storage

Store at 4 ° C for 6 months or freeze at -80 ° C for 1 year

Use

This product is provided for LABORATORY RESEARCH USE ONLY. It is not for diagnostic or therapeutic use.