Hello,
I would like to isolate total protein for phosphoproteomics using the Qiagen Tissue Lyser II (that we have in our department) for disruption of yeast lysis with glass beads. However, Qiagen did not measure the time that these adapters can keep the samples cold. They can be put in -80°C two hours before use, but how much does the bead beating heat the samples?
In the protocol for RNA extraction they say 5 minutes and there is no mention of pre-cooling the adapters. Normally, I know that people perform the lysis in a cold room with a bead beater. However, we don't have a bead beater that we can put in the cold room and I would prefer not to buy a new one. Also, I am guessing that the adapters pre-cooled at -80°C should be able to keep the samples cold for at least 5-10 minutes?
It would be nice to hear from someone with experience with pre-cooled adapters before I buy them.
Thanks in advance!
Hi, we have that same tissue Lyser.
I have homogenized lung tissues for protein analyses.
I have colleagues that also have used it for RNA extraction.
So far, for my protocol, what I do is I cool down the tubes where I will put my sample to homogenize with the beads in it already by placing the tubes on ice or in a fridge.
There's a protocol in Qiagen website that says the Hz and time you use homogenize your samples for protein extraction.. I can't remember which of all the manuals had it, but if you go to the website and look for protein extraction I am sure you will find it.
As for my personal experience, setting the tissue lyzer at 2 min 20Hz works fine. And I do this 2 times, because I need to change the position of the plate in the machine since one side gets more "shaken" than the other one. So to make sure all my samples are well homogenized and disrupted, I do 2min*2Hz , 2 times.
Then I make sure the Centrifuge I will use to spin down the tubes and obtain my supernatants is at 4 °C. With that so far I have been able to measure cytokines from my tissue samples as well as total proteins with no problem.
Just keep everything your using as cool as possible, and homogenize only a few samples at a time so you make sure they won't get too warm in the process.
Hope this helps
Hi Ivon,
Thanks for your reply. Of the samples are kept on ice before putting them in the adapters. I am thinking to run the bead beating with 2x24 adapters for eppendorf 2ml tubes or fastprep tubes and wondering if they can keep the samples cold, at least for 5 minutes. If you work with 2x2minutes with pre-cooled adapters it should be fine I think. Do you pre-cool the adapters in the -80°C freezer?
Thanks,
Sylvester
Yes samples are kept in wet ice.
The tubes for the bead beater also are kept in ice before and after you put the samples inside.
The plastic racks adapters are stored for 20 min in -20 freezer (i do it while I wait my tissues). That will keep the samples cool enough throughout the homogenization procedure.
I do not use lower temperatures because that freeze the tissue sample too much and it can't be homogenized well by the beads.
Hope this helps
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