The first step in most Western blotting experiments is lysing your cells to extract protein. You need to break open your cells in order to be able to isolate the proteins, and you need to do this with the least degradation and the most reproducibility possible. Depending on what your starting material is, there are a variety of options for lysing your samples to extract total protein.
Liquid nitrogen grinding
Grinding samples in liquid nitrogen is a simple, yet work-intensive way to extract total protein. This technique involves the use of a mortar and pestle, with a generous dose of liquid nitrogen. You use brute force (elbow grease!) to grind and pulverize the samples, and keep the bowl of the mortar topped up with liquid nitrogen to ensure that the samples will stay super cold (i.e. to protect them from degradation). The liquid nitrogen also makes the cell structure more brittle and easier to break. This technique is commonly used to break down plant tissue, as well as frozen cell pellets (especially yeast). The main drawbacks are that it can be messy and dangerous (due to copious amounts of liquid nitrogen on the loose), and physically demanding (for those of us with no measurable upper body strength!).
Sonication uses high intensity sound waves to disrupt cells, allowing you to extract proteins from tissue that has broken open. A sonicator typically comes with two differently sized tips to help you grind large amounts of samples in wide-mouthed tubes, or small samples in eppendorfs. Sonication works with varying efficiency, depending on the cell type. For instance, cells walls can be a lot more resistant to sonication disruption than cell membranes alone. For plant cells or diatoms, a “soni-thaw” method often works well (submerging the sonication tip in flash frozen samples and sonicating until they reach the consistency of a green slushie). The major advantage of sonication is its adaptability to different sample volumes and ease of use. However, you have to be very careful not to overheat your samples, so it is necessary to cool your tubes on ice between each sonication cycle, which can really eat up time.
Bead beaters are great for handling large volumes of cells. These apparatuses look like kitchen equipment, along the lines of a Cuisinart mixer without the blade. To break open cells in a bead beater, you fill the bowl with beads, your cell slurry, and protease inhibitors. Like sonication, bead beating works quickly and simply. However, since the bead beater motor is so powerful, it heats up the sample quickly, so it’s always a good idea to do this in a cold room and allow the whole apparatus to cool between beating cycles – make sure to wear a sweater, because you’ll be hanging out in the cold room all day!
Depending on your organism, you may find that buffers are the simplest way to break open your cells for protein extraction. Examples include Y-PER for yeast and RIPA buffer for mammalian cells. These buffers work chemically to break cells open, eliminating the need for mechanical means of cell disruption. Sometimes you can combine a lysis buffer with mechanical disruption for enhanced breakage, although mammalian cells are usually fragile enough that just buffer will do. Buffers are great because they simplify protein extraction: you can do it on your bench, and you don’t need any specialized equipment. Many people find that they get more consistent extraction too, because there is less variation in handling. The drawback, of course, is that handling samples at room temperature can lead to protein degradation, so it’s a good idea to at least keep your tubes on ice, and maybe even do the whole procedure in the cold room. Lysis buffers can also be more expensive than the more mechanical procedures.
It’s a good idea to always do a “test run” with a new species to optimize lysis conditions. For instance, try exposing cells to increasing levels of disruption (ex: 1x-8x 30 second runs on the bead-beater or 1x-4x soni-thaw rounds), or incubating in the lysis buffer for varying lengths of time. You can then determine the total protein quantity extracted and assay for degradation by western blotting. It sounds like a long process, but with practice you can develop a reliable and reproducible disruption protocol that maximizes protein release while minimizing degradation.
For more information on cell lysis and protein extraction methods, check out this video from Agrisera.
In the last post I showed you how to make an outline for your thesis in Word. You should now have a document outline with a list of headings for your sections (maybe even a few sections filled in if you were feeling motivated to make a start!). From here, we can move on to: […]
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