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Cell lysis is the breaking down of the cell membrane and the separation of proteins from the non-soluble parts of the cell. Lysate buffers contain different detergents that help to release soluble proteins. Dependent on the location of the protein of interest, a different lysate buffer is needed to obtain a high yield and purity of the protein.
Use protease inhibitors in the lysis buffer
The concentration of proteinase inhibitor(s) should be 1.5–2 times that used for Western blotting lysates. Proteintech usually uses RIPA buffer (Table 1), which enables efficient cell lysis and protein solubilization, while avoiding protein degradation and interference with the protein’s immunoreactivity and biological activity.
Optimize your lysate concentration
Commonly used amounts for IP: 0.2–0.5 ml lysate contain 1–4 mg total protein. Measure the total protein amount by protein assay, such as Bradford or BCA assay.
High concentrations of detergent interfere with IP
Try to lyse cells with a small volume of RIPA and then dilute the lysates with PBS (Table 2) to the final volume. Depending on the experiment, the detergent strength is crucial for the IP results (e.g., Triton-X, Tween, SDS, CHAPS).
Keep cells/tissue on ice and use ice-cold buffers.
|50mM Tris HCL, pH 7.4 (1 M stock)|
|1% Triton X-100 or NP-40|
|0.5% Sodium deoxylcholate|
|1mM EDTA (0.5 M stock)|
|Add ddH20 to the final volume|
|Add PMSF to a final concentration of 1mM and any other protease inhibitors immediately before use.|
|1 x PBS|
|137 mM NaCl|
|Adjust pH to 7.4|
|Add ddH20 to the final volume|
Pre-cool a refrigerated centrifuge to 4°C. Pellet the cultured cells by centrifugation at 4°C, for 5 minutes at 2000 rpm.
Wash 3 times with ice-cold 1X PBS and then add cold RIPA buffer with protease inhibitors.
Add 100 μl RIPA buffer for approximately every 10⁶ cells present in the pellet (count cells before centrifugation).
Reduce the volume of RIPA buffer accordingly if a higher protein concentration is required.
Vortex to mix and keep on ice for 30 minutes. Vortex occasionally.
Dissect the tissue of interest and wash briefly with cold 1X PBS.
Cut the tissue into smaller pieces while keeping it on ice.
Transfer the tissue to a homogenizer and add RIPA buffer with protease inhibitors.
Add 500 μl RIPA buffer for approximately every 20–50 mg of tissue.
Homogenize thoroughly and keep the sample on ice for 30 minutes. Vortex occasionally.
Sonication: high power, 1–2 minutes, then keep the sample on ice for 20 minutes.
Centrifuge at 10,000 rpm for 20 minutes at 4°C to pellet cell debris.
Transfer the supernatant to a fresh microfuge tube without disturbing the pellet. Determine protein concentration by protein assay, such as Bradford or BCA assay.
Samples can be frozen at -80°C for long-term storage, or be used for immediate immunoprecipitation.
Pre-clearing is a step that decreases binding of non-specific proteins, lipids, carbohydrates, or nucleic acids. This step is performed by incubation of the lysate with the solid support (e.g., agarose or magnetic beads) in the absence of the capture antibody.
Resuspend Protein A or G agarose bead slurry by gently vortexing the storage bottle.
Transfer beads and wash with 1X PBS 4~5 times before use. Quickly add 50 μl of 50% bead slurry per 1–4 mg of total protein to the microfuge tube containing the lysate.
Carefully cut the end of your pipette tip to facilitate pipetting and homogenization.
Incubate on a rotary mixer for 30 minutes at 4°C.
Centrifuge at 1000 rpm for 3 minutes at 4°C and transfer the supernatant to a fresh tube.