Bead Beating: A Primer (continued)
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Since many researchers use commercial kits for the harvesting of proteins and nucleic acids from samples, the protocols listed above provide no specific details about homogenization buffers. In most cases, the first buffer used for isolating DNA or RNA can be used as the homogenization buffer. Though fewer commercial kits are available for protein isolation, the same is essentially true. For those researchers on a tight budget or in a spot (e.g., it is a weekend and you need that molecule), here are a couple of basic homogenization buffer recipes and related protocols. Some reagents used in these protocols are dangerous, so exercise caution when using them.
Proteins are typically isolated as active molecules or in a denatured state. For best yields of native proteins, homogenization buffers should contain protease inhibitors, and the process should be done at 4°C. The final homogenate should also be refrigerated, as freezing of protein can lead to its denaturation. Alternatively, proteins can be isolated using buffers containing detergents such as SDS. The detergent denatures the proteins, including proteases, and prevents enzymatic breakdown. It must be noted that homogenization buffers with detergent will foam, which can hinder the movement and effectiveness of grinding beads and balls during beating.
Native Homogenization Buffer: 100 mM Tris, pH 7.6, 20 mM glutathione (DTT or 2-mercaptoethanol can be used), 5% sucrose, 2 mM EDTA (ethylenediaminetetraacetic acid), 1 mM PMSF (phenylmethanesulfonyl fluoride [this is toxic]), 5 µM Pepstatin A. Many different protease inhibitors can be added to homogenization buffers, many of which are not readily available. EDTA inhibits many metallo-proteases while PMSF acts against many serine and cysteine proteases. At a minimum, EDTA and PMSF should be included in the buffer. If the protein solution is to be used for assays involving formazan dyes, leave out the glutathione.
|1.||Add sample, buffer, and beads/balls to the disruption vessel. Keep cold until processing.|
|2.||Fix the homogenization vessel into the homogenizer and bead beat. Multiple short processing times can be used if excessive heat is a concern.|
|3.||Remove the vessels from the homogenizer and centrifuge the contents at high speed. This will remove insoluble cellular debris from the homogenate.|
|4.||Transfer the supernatant to a clean tube. Refrigerate the sample lysate until needed. Do not freeze the solution if there are concerns about activity loss due to denaturation.|
Denaturation Buffer: 50 mM Tris, pH 7.5, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecylsulfate.
|1.||Place sample, buffer, and grinding ball in vessel. Small beads may not work well with this buffer due to foaming.|
|2.||Process samples in a homogenizer. Heat buildup is not an issue as the buffer is denaturing.|
|3.||Centrifuge the homogenate to pellet insoluble debris. Transfer cleared supernatant to a clean tube.|
|4.||Sample can be refrigerated or stored frozen.|