Branson Ultrasonics Corp.
Digital Sonifier S-450D
2601-04
To overall goal of this research was to study protein localization to the flagellar membrane. The specific aim was to study the association of a specific protein with detergent resistant membranes. This product was chosen due to the availability within the department.
To sonicate cells
Trypanosoma cruzi cells
T. cruzi epimastigote cells were washed with PBS and incubated on ice in hypotonic lysis buffer for 10 min. Lysates were sonicated at 50% maximum output for two 5-s pulses with a 1-min cooling on ice between bursts using a Branson Digital Sonifier. Samples were pre-cleared at 10,000 × g for 10 min to remove unbroken cells and nuclei. Clarified supernatants were then centrifuged at 100,000 × g at 4 °C for 1 h using a Beckman TLA100.3 rotor. Supernatants were precipitated with acetone.
Check for sucessful lysis on the light microcope before proceeding
Transfectants were lysed by hypotonic sonication and fractionated into a cytosolic and membrane fraction by high speed centrifugation. Although endogenous protein (lower molecular weight bend) of interest partitioned with the membrane fraction, the tagged mutants (higher molecular weight band on the gel) were both found exclusively in the cytosolic fraction. As expected, known cytosolic (C) and membranous (M) proteins associated predominantly with cytosolic and membrane fractions, respectively.
Quick pulse reaction, adjustable power
None
Ease of use
Not widely available
This sonicator was successful at lysing cells thus getting the job done.