UV Crosslinking Protocol
UV-Cross-linking in decapping extracts
- Set up the reaction in a 1.7ml tube:
7-8μl extract (15-20 mg/ml) (or recombinant protein)
1μl 20 mM MgCl2 (optional)
1μl 1 mM ATP (optional)
1μl hot RNA (50-100 kcpm/μl)
- Incubate at 30 ° C for 5-10 min.
- Transfer to a small microtiter dish at 4 ° C.
Cross-link with 254nm UV light for 10 min. The light should
be 2-3 cm from the dish. Make sure it all lines up well.
- Transfer to a 1.7 ml tube containing 2μl RNAse
A (10mg/ml) and incubate at 37 ° C for 10 min.
- Add an equal volume of 2 x SDS loading dye. Boil
4 min and load on 0.8 mm thick SDS/PA gel.
- Transfer to Whatman 3MM paper and vacuum dry for
- Expose to film at -80 ° C.
UV-cross-linking to radio-labeled RNAs.
- Incubate in vitro transcribed RNAs with the extract
or protein of interest for 5 min at 30 °C. A 10
ml reaction volume is ideal. Buffer conditions can
50-100 µg of extract or 100ng-1µg of recombinant
50-100 kcpm of 32P-labeled RNA (internally labeled
- Transfer each reaction to a 50 well microtiter dish on ice.
Generally, the UV light will efficiently radiate
only two rows so bear this in mind when picking which wells to
- Line the UV light up with the wells and switch on for 10
min. The light should be 1-2 cm from the dish. Make sure to use
the short wave
- Transfer the reactions back to 1.7ml tubes containing 1.5µl
of RNAse A (and 0.3µl RNAse One if the substrate contains
poly(A)). Incubate at 37 °C for 10 min to degrade
the input RNA.
- Add an equal volume of 2 x SDS loading dye. Boil and load
on an SDS-gel. Dry the gel and expose.
Immunoprecipitation of cross-linked proteins.
Follow the protocol described above up to step 4.
- Add 400µl of NET-2 buffer and 1-4µl of antibody
to the reaction. Incubate on ice for 1 hour.
- Spin for 2 min to pellet any precipitated proteins. Transfer
the supernatant to a fresh tube.
- Add 20µl of a 50% slurry of protein A sepharose (pre-washed
in NET-2). Incubate with gentle rocking at 4 °C for 20 min.
- Spin down the protein-A beads and remove the supernatant.
- Wash the pellet 3-5 times with RIPA buffer.
- Resuspend the pellet in 25µl of 2 x SDS-loading dye.
Boil and load on SDS-gel.
50 mM Tris-Cl (pH7.6)
150 mM NaCl 0
50mM Tris-Cl (pH7.6)
Partial V8 proteolysis of cross-linked
This protocol allows comparison of cross-linked proteins in extracts
with recombinant proteins or allows bands of the same size to be
Follow the protocol described above, up to step 5. Either load
4 lanes for each sample or use an extra wide well comb. It is important
to use a 0.75mm thick gel for this step. Do not dry the gel!
- Expose the wet gel to film or phosphorimager, and excise
the band of interest. Insert each gel slice into the well of a
1.5 mm thick 15% SDS gel with a long stacking gel (7-8cm).
- Overlay each gel slice with 40µl of 2xSDS loading dye
containing V8 protease. There should be four lanes for each sample
so add increasing amounts of protease – e.g. 0µg, 0.05µg,
0.5µg and 5µg.
- Run the dye two-thirds through the stacking gel. Turn off
the power for 30 min to allow the digestion to proceed.
- Turn the power back on and run the dye front to the bottom
of the gel as normal. Dry and expose.