I have dissected out mouse hippocampus and want to homogenize it for analysis of phosphorylated and aggregated tau. We have dounce homogenizers but they feel too big for such small tissues. We also have a TissueLyser from Qiagen (normally used for RNA-prep) which uses a bead to disrupt the tissue, but I have been told that too strong mechanical disruption can destroy the large aggregates. Are there any do's and don't-s for this type of sample preparation?
Harald, What method are you going to use for the analysis, Western blotting? I have homogenized hippocampi using thermoscientific powergen as well as direct probe sonication. Either way I was able to do western blotting.
Hi Harald,
Did you try some kind of micro - homogenizer e.g.
http://www.wilmad-labglass.com/uploadedFiles/Main_Site/Content/Literature_Center/Micro-Vial%20Homogenizer%20Web%20Binder.pdf
or
http://www.thomassci.com/Equipment/Homogenizers/_/72628210-F46B-44F2-AF74-6DB77AC18825?q=Tissue%20Homogenizer
I used the second one for hippocampus lysis, and it was ok.
Regards
Monika
Hi and thank you both for your answers! I will primarily do ELISA to measure total tau, phosphorylated tau and oligomeric/aggregated tau. The micro-homogenizers look very suitable. Have you both run analysis of tau oligomers (or similar) on you homogenates?
Harald, I haven't done tau or any other phosphorylated proteins. I immunoblotted for dopamine receptors.
Hi Harald , In our lab we analyzed phosphorylation and other PTM modifications in hippocampus homogenates.
Regards
as suggested by Monica glass or platic pestle homogenizers work fine for western of tau and its aggregates - make sure to beep it all cold & add proteinase & phosphatase inhibitors cfr link attached
http://onlinelibrary.wiley.com.kuleuven.ezproxy.kuleuven.be/doi/10.1111/ejn.12595/full
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