preliminary comment:The following text is taken from my Materials and Methods, so please excuse the raw way of writing.
Aim of the technique:
This technic allows for the visualization of how well a known RNA interacts with a know protein in the presence of its partners (the partners of the RNA binding protein of course).
Brief introduction:
- You incubate a radioactive RNA with a protein extract
- specifically crosslink the RNA-protein interaction with UV light (254 nm)
- digest unbound parts of the RNA so that only a few radioactive and non-radioactive nucleotides remain bound to the protein (little variation in the size of the protein makes it easier to be identified while running the samples on a gel). Alternately, you can confirm the protein's identity by immunoblotting.
-Immunoprecipitate your protein of interest, crosslinked to the radioactive nucleotides and identify it by running samples on a SDS-PAGE and autoradiography.
Enough talking, here is the protocol:
required products:
-nuclease-free water (prepare DEPC-treated water: incubate 1L of water with 0.1% v/v of diethyl pyrocarbonate overnight at 37°C with vigorous stirring and autoclave it)
-Dynabeads protein A (also test protein G coupled beads which may work better depending on the class of antibody you want to immobilize)
- PBS,
- molecular grade BSA powder
- Tris-HCl pH 7.9,
- NaCl,
- Nonidet P40
- radioactive body-labeled RNA (obtained by in vitro transcription with alpha P32 UTP)
Avoid 5'end labeled RNA if your not sure that your protein binds on the first nucleotide of your RNA)
tips before starting:
-don't forget to load the input lane corresponding to each immunoprecipitation you present (composed of the radioactive RNA+ protein extract treated with RNase A)
-don't forget to do a negative control (isotype control antibody: same class as your antibody, but not raised against any antigen). A negative control without antibody bound to the beads won't work here due to physical properties of the Dynabeads, which is also the reason why I tried to decrease non-specific binding of RNA and proteins to the beads by pre-blocking them TWICE! with BSA.
-It goes without saying that all solutions are better working when prepared freshly and that all solutions need to be prepared in nuclease-free water.
protocol:
200,000 cpms of a radioactively body-labeled RNA were washed in 70% ethanol, dried and resuspended in 16 µL water. To this solution, 10 µg of yeast tRNAs and 20 µL of a nuclear extract prepared from HeLa cells (~30 µg/µL)were added. This mixture was incubated for 15 min on ice, exposed to UV light for 15 min on ice (UV Stratalinker™ 2400, lambda=254 nm, Stratagene), and treated with Rnase A at a 1 µg/µL final concentration for 15 min at room temperature. Meanwhile, 20 µL of a 50% suspension of Dynabeads coupled to protein A (Invitrogen) were washed once with 1 mL PBS and pre-blocked 30 min at room temperature with 0.2% BSA complemented PBS buffer under gentle rotation (16 rpms). The resin was washed three times with 1 mL of NET-2 buffer (50mM Tris.HCl pH 7.9, 100 mM NaCl, 0.05% NP40, 0.5 mM DTT) and pre-blocked again for 1 hr at 4°C in 5% BSA complemented PBS. Following three successive washes in NET-2 buffer, the beads were resuspended in a final volume of 42.5 µL of NET-2 buffer and incubated for 1 hr at room temperature with 3 µg of antibody, under gentle rotation. Antibody-coated beads were washed three times in 1 mL of NET-2 buffer and incubated for 1 hr at 4°C with to the former mixture of nuclear extract with radioactive RNA and tRNA, under gentle rotation. The unbound proteins were washed from the beads three times with 1 mL of NET2 buffer and elution occurred by addition of 12 µL of 1X sample solution and heating 5 min at 95°C.
Don't hesitate to comment or contact me for troubleshooting.
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