Actin expression in protein extracted from tissue

Kamalian, Laleh via methods%40net.bio.net (by L.Kamalian from liverpool.ac.uk)
Fri Apr 3 05:46:12 EST 2009


Hi.
Thanks to every body who responded.  The reason I kept the tissue in RNAlater was that they were supposed to be used for RNA extranction.  However my supervisor asked me to extract protein out of them.  The information leaflet came with the solution claims that the solution doesn't touch the protein and they are safe.  I also double check this with their costomer services and they said the same thing.  The CellLytic-M is the lysis buffer that we have been using to extract protein from our cell-lines and it has been serving us well.  The centrifuge speed was 13000 rpm.  Above all this method of extraction was previously done by one of my colleagues and worked very well, it gave a single band of b-actin as the normalizer.  I followed her instructions exactly.  I tried mouse monoclonal anti GAPDH yesterday and it also gave me nonspecific bands, although the actual band ( according to the size) was much better than the actin's.   So is there any further thoughts?
Thanks
L.


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