1 reply to this topic

[Tfal]

Hi,

so I started working with small mouse muscles (soleus, EDL, etc).
For other tissues such as liver or bigger muscles (gastrocs, quads) or rat muscles I would use 50-100mg of tissue with 10 ul of lysate buffer/ mg with a bead shaker and it would work fine.
But those small mouse muscles weigh 4-20 mg and with 10 ul of lysate buffer/ mg with a bead shaker, the muscles don't get homogenized. The muscles stick on the eppendorf out of the buffer and are spared by the beads.

I started using a blade grinder on polytron with much more buffer 40-100ul / mg. It seems to work OK, although sometimes the muscle stick on the outside rod or some conjunctive tissue gets stuck in the blades.

But I wonder if the two methods are equivalent, especially when using more buffer. How about a Mini-Potter or glass-on-glass tube.
And should I sonicate afterwards. What does it change.

By the way I use Triton x-100 and DTT in my buffer.

thnx
T

[gfischer]

I assume you're homogenizing for isolation of protein.  If so, I'd recommend using either a glass conical or a dounce homogenizer.  In my experience, you get better yields with these than with motorized systems.  In small volumes, the heat generated from sonication can damage some proteins.  I'd use somewhere between .5 and 1mL lysis buffer, depending on what sort of concentration you need.