Approaches for the Identification and Characterization of RNA–Protein Interactions - Biophysical Approaches to Translational Control of Gene Expression - page 206

6. Add an equal volume of 2× SDS loading buffer, mix the samples by vortexing,
heat to 100 °C for 2 min, and after brief centrifugation (to remove any precipi-
tated proteins) electrophorese on a 10% SDS-polyacrylamide gel.
7. Run the gel until suf fi cient resolution has been obtained.
8. Transfer the gel to Whatman filter paper, cover with plastic wrap, and dry with a
gel-drying apparatus.
9. The dried gel should be exposed to the phosphor screen and analyzed by
phosphorimaging.

10.4.2.3 Notes

1. We typically use 10-15 mL total reaction volumes in the protocol. While amounts
of individual components need to be determined empirically, 50-100 m g of
extract (or 100 ng to 1 mg of recombinant protein) and 50-100 K cpm of
32 P-labeled RNA are good starting points.
2. Reaction mixtures can also be immunoprecipitated following irradiation and
RNAse treatment to confirm the identity of the cross-linked protein. The protocol
used for this is similar to that found in Sect. 10.2.3.5 , steps 1-4. Following wash-
ing of the beads, simply resuspend the sample in 2× SDS loading buffer and
follow steps 6-9 above.

10.4.3 In Vitro Af fi nity Puri fi cation of RNA Binding Proteins

A common strategy to uncover the identity of an RNA binding protein is to purify it
by taking advantage of its ability to specifically interact with an RNA substrate.
Following purification, the protein is identified using mass spectrometry. The
method described below focuses on a small scale purification using short pieces of
RNA that will likely interact with a single or small number of proteins. It is impor-
tant to appreciate that a similar approach can also be applied to larger RNAs and
global proteomic analysis using Ribo-Trap technology (Beach and Keene 2010 ) .

10.4.3.1 Specialized Materials

1. Biotinylated speci fi c and nonspeci fi c RNA target oligomers (IDT).
2. Cellular extracts (prepared as described for example in Sokoloski et al. 2008 ) .
3. Streptavidin agarose resin (Thermo Scienti fi c).

Buffers :

1 . Buffer D : 20 mM HEPES-KOH, 20% glycerol, 100 mM KCl, 0.2 mM EDTA,
1 mM DTT.
2 . HSCB : 400 mM NaCl, 25 mM Tris-Cl, pH 7.6, 0.1% SDS.