Currently, we use surgical scissors to cut the tissue (previously frozen at -80 degrees) while in a homogenization buffer. Then we place a metal bead in the tube and employ bead beating homogenization at 40-50 osc/min for 1 minute 6 times. We then centrifuge at 10,000g for 15 minutes and collect the super. Is this excessive? Coomassie blue stain shows protein transfer to membrane and bio rad protein assay shows high protein concentration; however, there are inconsistent results from probing. Is it possible that the target protein was denatured in the homogenization process?
It's a few years when we tested a bead based homogenizer with human and mouse skeletal muscle samples. Surprisingly, we were not able to detect expected phosphorylations of proteins involved in insulin signaling. We resorted to a rotor-stator device then and everything was fine.
Other groups had no problems with (other) bead based machines, so it might be worth digging around a bit in the market.
I prefer tungsten carbide beads instead of metal and apply cold treatment between each pulses...You may lose your phosphoproteome by nonspecific binding and generated heat during homogenization...
Protease cocktails are. also needed to inhibit potential proteolytic activities....
Homogenization buffer composition is important to be known here in...what kind of lysis buffer composition do you have and did you take into account the above details...
İsmail Emir Akyildiz Thank you for the response. Inhibitors were used (10ul of protease and phosphatase each per 1ml of lysis buffer) but I'm thinking the heat from the prolonged homogenization severely affected my samples. I applied cold treatments between pulses but the pulses were 1 minute sometimes 2 at a very high oscillation speed.
As for the buffer, we used RIPA.
Used RIPA, 40 Hz, 4,800 rpm, for 1-min pulses, three times, with a 30-s rest on ice between pulses, using carbide or zirconia beads...
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