Ultrasonic Lysis for Western Blotting

• The western blot is an analytical procedure for the detection of specific proteins in a sample of tissue homogenate or cell extract.
• To run a Western blot or to measure enzyme activity, many assays require access to the materials (e.g. proteins, DNA, subcellular fragments) entrapped in the cell.
• Sonication is a reliable and easy-to-handle method for controlled cell disruption and lysis.

Ultrasonic Cell Disruption

Protein extraction from tissues and cultured cells is the first step for many biological, biochemical and analytical techniques (PAGE, Western blotting, ELISA, mass spectrometry, etc.) or protein purification. To obtain a high protein yield, the cell material and tissue must be efficiently disrupted/ lysed. Whether plant cell or animal tissue, sonication is the method to prepare your cell lysate easy and fast.

Advantages of Sonication

• fast & efficient
• easy operation
• high protein yield
• reproducible/ repeatable
• precisely controlled
• scalable

Immunoprecipitation Protocol For Western Immunoblotting

A. Reagents

For the preparation of the solutions, use purified water such as Milli-Q.

• 1X Phosphate Buffered Saline (PBS)
• 1X Cell Lysis Buffer: 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
  Important: Add 1 mM PMSF immediately prior to use.
• 15 μl Protein A+15 μl Protein G is sufficient for IP, but it may depend on your primary antibody and sample volume. You can also use pre-mixed protein A/ G agarose (e.g. Protein A for rabbit IgG pull down and Protein G for mouse IgG pull down)
• 3X SDS Sample Buffer: 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue

B. Preparation of Cell Lysates

• Harvest the cells. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
• Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate the plates on ice for 5 minutes.
• Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
• Sonicate twice for 10 seconds in ice-cold immunoprecipitation buffer (IP buffer: 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.1% NP-40 and the protease inhibitor mix). For sonication, the VialTweeter or a probe ultrasonicator such as the UP100H or UP200Ht are most suitable.
• Centrifuge the lysates at 15,000 g for 10 minutes at 4°C.
• Transfer the supernatant to a new tube. (If necessary, lysate can be stored at –80°C.)
• Add the primary antibody to the supernatant. The supernatant with the primary antibody is incubated for 1 h at 4°C under light agitation. Primary antibody is typically added in an amount 10x more concentrated than is used for western blotting. (You may start with 1μg per 100μL.)
• The supernatant is then incubated further with the mixture of equal amounts of Protein A-agarose (Invitrogen) and Protein G agarose for another 1 h.
• Wash the agarose pellets three times with IP buffer. Afterwards, extract the bound proteins with the SDS-PAGE loading buffer by heating at 95°C for 5 minutes.

C. Immunoprecipitation

• Take 200 μl cell lysate and add primary antibody. Incubate with gentle rocking overnight at 4°C.
• Add either protein A or G agarose beads (20 μl of 50% bead slurry). Incubate with gentle rocking for 1–3 hours at 4°C.
• Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes.
• Resuspend the pellet with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 seconds.
• Heat the sample to 95–100°C for 2–5 minutes and microcentrifuge for 1 minute at 14,000 X g.
• Load the sample (15–30 μl) on SDS-PAGE gel (12–15%).
• Analyze sample by Western blotting.

Western Blot Analysis with the Sonicator UP50H

Following protocol using the probe-type sonicator UP50H was used in study of Kriebisch et al. (2011):
Total protein was isolated from MC3T3-E1 cells treated with 1,25(OH)₂D₃ (10⁻⁸ M) or vehicle. Cells were lysed with a buffer containing 50 mM Tris HCl, pH 8 (Sigma-Aldrich); 150 mM NaCl (Fisher Scientific); 0.1% sodium dodecyl sulfate (SDS) (Fisher Scientific); 1% IGEPAL CA-630 (Sigma-Aldrich) and 0.5% sodium deoxycholate (Merck). The cell lysate was sonicated for 2 × 10 s at cycle 1 and amplitude 80 with the UP50H Ultrasonic Processor (Hielscher, Ultrasound Technology, Teltow, Germany). Thereafter the material was centrifuged for 10 min at 14,000 rpm and the supernatant was used for Western blotting. Twenty-five μg of protein was boiled in sample buffer and reducing agent (Invitrogen) and subsequently separated by SDS-PAGE using 4–12% polyacrylamide gels (Invitrogen) and transferred to a nitrocellulose membrane (GE health care). The membrane was blocked for 1 h with TBS (10 mM Tris-HCl; pH 7.6; 150 mM NaCl) containing 1% casein (Sigma-Aldrich) and 1% Tris (1 M). After blocking, the membrane was incubated with slight agitation overnight at 4°C with the primary antibody (rabbit anti-human CBS 1/500, developed in the lab of Prof. R. Banerjee, Ann Arbor, MI, USA). Incubation with a horseradish peroxidase (HPR)-conjugated secondary antibody (Dako) was performed for 1 h at room temperature. All blots were developed by enhanced chemiluminescence (Perkin Elmer).
 

Click here to read more about best practices for ultrasonic cell lysis and extraction, lysate preparation and recommendations for process improvements!
 
The table below gives you an indication of the approximate processing capacity of our lab-size ultrasonicators:

Literature / References