Proper sample preparation is key to successfully detecting your protein of interest by Western blot. Sample preparation will vary depending on the type of cell or tissue, protein, and organism. For extraction from mammalian cells, membrane proteins, such as multipass G-protein coupled receptors, require more stringent lysis conditions than cytosolic or nuclear proteins. Detection of nuclear proteins requires thorough lysis but can result in a viscous lysate due to DNA released from the nucleus.
There are a variety of ways to lyse cells and homogenize a sample for protein detection by Western blot. One method, sonication, uses ultrasonic frequencies of 20 kHz or greater to disrupt cell membranes and ensure complete cell lysis. While most homogenization techniques are capable of cell lysis and homogenization, sonicators are particularly efficient. Where sonication stands out from other methods is its ability to shear chromatin and DNA in small sample volumes. DNA released from lysed nuclei can create a viscous sample that interferes with sample preparation and gel loading. Sonication shears DNA, decreasing viscosity, homogenizing the sample, and releasing DNA-bound proteins.
Benefits of Sonication:
- Decreases viscosity. A highly viscous solution is difficult to load by pipette. Even after brief centrifugation, genomic DNA does not pellet well, and it is difficult to remove the supernatant without disrupting the genomic DNA pellet.
- It minimizes non-specific binding to the DNA and releases DNA-binding proteins. If your protein(s) of interest are nuclear and/or known DNA-binding proteins, this step is critical to ensure they are represented in your Western blot sample.
- Sonication creates a homogenous sample, allowing for loading consistency across lanes. This is critical for quantification of bands.
- Shears DNA that can interfere with protein separation by gel electrophoresis.
- Increases signal. Sonication can significantly improve detection as protein extraction is much more efficient. This is especially true for nuclear or membrane proteins.
Sonication Best Practices
One downside to sonication, however, is that samples can become hot, which can degrade proteins. Samples can also become foamy with extended sonication or if the probe is moved in and out of the sample volume, denaturing proteins. While any homogenization method will cause some protein denaturation, to minimize this impact, best practices for probe sonication include:
- Use short five-second pulses to avoid oversonicating. Allow cells to rest in between each pulse for at least a 1:1 ratio of pulse to rest time. Total sonication time depends on your cell or tissue type and should be optimized for protein extraction; protocols range from 10 seconds to two minutes.
- Keep samples cool during sonication. To avoid overheating, place samples on ice during sonication. At minimum, samples need to be cooled between pulses. Some probes come with or can be fitted with accessories for integrated cooling. Read our previous blog post, which discussed several ways to address heat accumulation during sonication.
- Use an appropriate amplitude. This will depend on the diameter of the probe you are using and the amplitude setting selected (displayed as a percentage). The greater the amplitude, the more intense the sonication. Amplitude is more important for determining the intensity of sonication than the power setting.
The Continuous Flow Attachment for Sonifier Ultrasonic Homogenizers has a built in cooling jacket that helps dissipate heat.
To protect yourself, make sure to wear ear protection or use an enclosure, such as the Acoustic Enclosure for the Sonifier or the Sound Enclosure for Q55 and Q125 Sonicators by Qsonica to decrease the decibel rating.
Resources:
https://www.cellsignal.com/learn-and-support/videos-and-webinars/should-you-sonicate-your-wb
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/cell-lysis-total-protein-extraction.html (Cell lysis buffers for varying sample and protein types)
