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Purification of RNA binding proteins by avidin agarose beads

2D gel electrophoresis

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2 replies to this topic

#1 kartiga natarajan

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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......

#2 neuron

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Posted 05 March 2013 - 03:17 AM

Hey did you run 2D gel? Ideally it should work...Posted Image

Edited by neuron, 05 March 2013 - 03:18 AM.


#3 mdfenko

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Posted 05 March 2013 - 05:39 AM

you may have left the rehydrated strip too wet but it may be okay. you can stain the strip to determine if focusing went well.

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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