Materials and Reagents

96-well Microplate Nunc maxisorp White (Thermo Fisher Scientific, Nunc, catalog number: 436110 )
Recombinant 6x HIS tagged protein
Streptavidin (New England Biolabs, catalog number: N7021S )
NaHCO₃
Phosphate buffered saline (PBS) (Life Technologies, Gibco^®, catalog number: 10010031 )
Tween 20
BSA
Biotinylated RNA
1 M Tris (pH 7.5)
NaCl
Yeast tRNA (Life Technologies, Applied Biosystems^®, catalog number: AM7119 )
6x His mAb-HRP Conjugate (Clontech, catalog number: 631210 )
ECL Peroxidase Substrate Solution A and B (Pierce Antibodies, catalog number: 32106 )
Water used for all solution is RNAase free
q-PCR tape (R&D systems, catalog number: DY992 )
RiboLoc RNAase Inhibitor (Thermo Fisher Scientific, catalog number: EO0381 )
RNA 3' End Biotinylation Kit (Pierce Antibodies, catalog number: 20160 )
Binding buffer (see Recipes)
PBS-T (see Recipes)

Equipment

Luminescence Plate reader (BMG LABTECH, FLUOstar OPTIMA)
Thermocycler
Centrifuges

Procedure

Coated 96-well plates with 50 μl/well of streptavidin (100 μg/ml in 0.1 M NaHCO₃).
Incubate overnight at 4 °C. Seal the wells with q-PCR tape.
Wash plates six times with 200 μl PBS-T (there is no need to incubate for washing).
Block with 50 μl/well of PBS containing 3% BSA and 0.1 μg/ml streptavidin.
Incubate overnight at 4 °C or 7 h at RT. Seal the wells with q-PCR tape (for my experience, in this step overnight or at least 7 h RT incubation is needed as less time causes high noise).
Mix in pcr tubes biotinylated RNA (5 pmol) and different amounts of protein (0-500 ng) in binding buffer. The final volume in each tube is 50 μl. The RNA was biotinylated using the RNA 3' End Biotinylation Kit.
Incubate the pcr tubes with the biotinylated RNA plus the protein, in a thermocycler for 30 min at 37 °C.
Wash plates four times with 200 μl PBS-T.
Transfer the mix (biotinylated RNA-protein) in to the plates (50 μl/well) and incubate 1 h at RT. Seal the wells with q-PCR tape (No need to rock to rock/shake the plate).
Wash plates six times with 200 μl/well PBS-T.
Add the 6x His mAB/HRP conjugate in PBS 1: 1,000 (50 μl/well) incubated for 1 h at RT. Seal the wells with q-PCR tape
Wash plates six times with 200 μl/well PBS-T.
Add 50 μl/well of a mix 1:1 ECL (25 μl of ECL1 and 25 μl of ECL2).
Spin the plate to avoid bubbles using an adaptor for a swing bucket centrifuge.
Read luminescence in the Labtech FLUOstar OPTIMA (MARS Software from labtech on Nunc maxisorp 96. 0.5 second interval).

Recipes

Binding buffer
50 mM Tris (pH 7.5)
150 mM NaCl
0.02 mg/ml yeast tRNA
0.2 mg/ml BSA
1.5 μl RiboLoc RNAase Inhibitor
RNAase free water
In a total volume of 50 μl per well
PBS-T
1x PBS
0.1% Tween 20

Acknowledgments

This protocol was adapted from Gajjar et al. (2012). This work was supported by INSERM, La Ligue Contre le Cancer, and RECAMO CZ.1.05/2.1.00/03.0101. M.M.C. was supported by JSPS, AXA Research Fund, and Fundacao para a Ciencia e Tecnologia of Portugal. M.G. was supported by a research fellowship from Indo-French Centre for the Promotion of Advanced Research (IFCPAR) and from the ARC.

References

Gajjar, M., Candeias, M. M., Malbert-Colas, L., Mazars, A., Fujita, J., Olivares-Illana, V. and Fahraeus, R. (2012). The p53 mRNA-Mdm2 interaction controls Mdm2 nuclear trafficking and is required for p53 activation following DNA damage. Cancer Cell 21(1): 25-35.
MacCallum, D. E. and Hupp, T. R. (1999). Induction of p53 protein as a marker for ionizing radiation exposure in vivo. Methods Mol Biol 113: 583-589.