Purification of RNA binding proteins by avidin agarose beads2D gel electrophoresis
Posted 19 February 2013 - 09:26 PM
I need to purify RNA binding proteins(RBP)...where i have prepared my biotin RNA by in-vitro transcription and allowing them to bind with cytoplasmic extracts...and i use avidin agarose beads to purify the proteins which bind to my biotinylated RNA... In case of running SDS, I have to boil those beads after purification in SDS Sample buffer for the breakage of biotin-avidin...But i need to run my RBP in 2D... So i suspended those beads in Urea rehydration buffer and i did 2D but during 1st dimension strips got swelled out....Do you guys have any idea regarding this?
Please help me out regarding this......
Posted 05 March 2013 - 03:17 AM
Edited by neuron, 05 March 2013 - 03:18 AM.
Posted 05 March 2013 - 05:39 AM
did you confirm that the urea buffer released the biotinylated sample from the avidin?
the denaturing conditions may separate the rna from the protein (do you want to do this?).
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