help: Northwestern blot assay

IJ Huang gr8bn at cc.usu.edu
Mon Jan 22 22:26:45 EST 1996


Dear netter
   I am trying to map a RNA binding domain of a RNA binding protein.  
Because this recombinant protein and its deletion mutants are quite 
insoluble both in E.coli and insect cells, I have difficulty to purify all 
of them.  I decided to do Northwestern blot assay to map the binding 
domain.  
    First, I separated the full-length and the deletions on SDS-PAGE, 
then transfered the proteins to a nitrocellulose membrane by either 
electrical blotting or by diffusion.  I tried to renature the proteins on 
membrane by incubating the membrane with 10 mM Tris, pH8, 50 mM NaCL, 0.1% 
Tx-100, and 1x Denhardt's reagent for 1 hr with 3 changes.  Then I added 32P-
RNA to the binding solution, washing, drying, and autoradiography.
   My problem is that the results were not reporducable.  Can anybody give 
me any suggestion?  Thanks in advance.

IJ Huang
Graduate Student
--------------------------------------------------------------------------
EMAIL:GR8BN at CC.USU.EDU              HTTP://CC.USU.EDU/~GR8BN/HUANG.HTML
PHONE: 1-808-797-2724               FAX: 1-801-797-1575



More information about the Methods mailing list