Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays.
We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin-agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect the formation of protein-RNA complexes. The EMSA that we describe avoids the use not only of radioactivity but also of neurotoxic acrylamide by using agarose as the gel matrix in which the free nucleic acid is separated from protein-nucleic acid complexes. After separation of free from complexed RNA in agarose, the RNA is electroblotted to positively charged nylon. The biotin-labeled RNA is readily bound by a streptavidin-alkaline phosphatase conjugate, allowing for very sensitive chemiluminescent detection ( approximately 0.1-1.0 fmol limit). Using our system, we were able to purify both known iron-responsive proteins (IRPs) from rat liver and assess their binding affinity to RNA containing the iron-responsive element (IRE) using the same batch of biotinylated RNA. We show data indicating that agarose is especially useful for cases when large complexes are formed, although smaller complexes are even better resolved.
Rodgers
J T
JT
Department of Chemistry, John Carroll University, 20700 North Park Boulevard, University Heights, Ohio 44118, USA.
Patel
P
P
Hennes
J L
JL
Bolognia
S L
SL
Mascotti
D P
DP
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001710:Biotin; D008863:Microspheres; D009696:Nucleic Acids / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D012685:Sepharose
D009696:Nucleic Acids; D011506:Proteins; D001710:Biotin; D012685:Sepharose