Tissue Protein Extraction: Tissue Homogenization using Urea-based Buffer and Bead Mill Homogenizers
J P Rose, M A Watson, B Schilling, Joanna Bons
Abstract
Tissue homogenization to isolate protein in preparation for downstream proteomic profiling.
Steps
Chill the adaptor sets of the bead mill homogenizer at -20°C.
Freshly prepare the lysis buffer as described in Table 1 and keep cold.
| A | B | C |
|---|---|---|
| Urea | 8 M | 4.8 mg |
| TEAB, pH 8 (1 M) | 200 mM | 2 mL |
| Nicotinamide (30 mM) | 3 mM | 1 mL |
| Sodium chloride (5 M) | 75 mM | 150 µL |
| HALT protease/phosphatase single-use inhibitor cocktail (100x) | 1 x | 100 µL |
| Trichostatin A (5 mM) | 1 μM | 2 µL |
| HPLC-grade water | N/A | N/A |
| Total | N/A | 10 mL |
Table 1. Lysis buffer composition for a final volume of 10 mL.
On dry ice, add the tissue specimen to a 2-mL microcentrifuge tube compatible with the bead mill homogenizer, then place the metallic bead into the tube. Add 500 µL of cold lysis buffer to the tissue specimen (add more lysis buffer to cover the tissue specimen, if necessary).
Place the tubes in the prechilled homogenizer adaptor sets, ensuring that the tubes are balanced between the two adaptors.
Homogenize the samples for 2 cycles at 24 Hz for 2 min each. If the tissue specimen is not fully homogenized, spin the samples briefly, transfer the homogenized lysate into a clean 1.5-mL microcentrifuge tube, add additional lysis buffer, and repeat the homogenization step. Two cycles of homogenization are typically enough to break up the tissue.
Remove the bead with a tweezer. In between each sample, rinse the tweezer with milliQ water, then HPLC-grade methanol, and dry thoroughly with a delicate task wiper.
Transfer the homogenized lysate into a clean 1.5-mL microcentrifuge tube or combine the homogenized lysates if needed, carefully avoiding bubbles and the separated fat layer at the top of the supernatant.
Centrifuge the homogenized lysate at 15,700 x g for 15 min at 4°C to clear the lysate.
Transfer the supernatant (clear lysate containing the soluble proteins) to a clean
1.5-mL microcentrifuge tube, carefully avoiding any liquid fat layer on top and
debris at the bottom of the tube.
Perform a bicinchoninic acid (BCA) protein assay to determine the protein concentration of the clear lysate with a proper dilution (typically 1:20 - 1:200 depending on the tissue type). According to the BCA results, transfer an aliquot of the sample into a clean 2-mL microcentrifuge tube for subsequent proteolytic digestion.
