Diagenode
Bioruptor Plus sonication device
B01020001
I'm interesting in understanding the molecular mechanism of hedgehog signaling pathway. In this experiment, my goal is to identify binding partners of a cilia localized protein in cultured cells by Mass Spectrometry. The first step in this process involves extraction of proteins from ~5 mg cell pellet. Efficient protein extraction and removal of DNA is critical for this initial step. Diagenode's Bioruptor was used for this process.
Protein extraction and DNA shearing
NIH-3T3 cells resuspended in cell lysis buffer
Cells were resuspended in a lysis buffer containing 1% NP-40, resuspended thoroughly, and then transferred to 2x50 ml tubes. Samples were subjected to sonication at 4 deg C with pulse cycles - 30sec ON and 30sec OFF, for 10 min.
Make sure to cool the system 30 min prior to the sanitation process.
Large scale protein-protein interaction assays require high quality protein with animal to no DNA contamination. Presence of DNA leads to viscous sample and affects affinity purification step and additional downstream processes. Sonication by Bioruptor shears DNA and eventually sheared DNA can be eliminated from the protein sample prep by ultracentrifugation. Bioruptor from Diagenode worked well for this purpose.
Easy to use
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We have tested so far for protein extraction and DNA shearing and it works very well for these two purposes.