I am trying to do the affinity purification of MS2-tagged mRNA by capturing it on GST-MCP (MS2 coat protein) immobilized on glutathione beads.
I used two approaches: transfecting the construct with my target MS2-tagged RNA and transfecting GST-MCP, then mixing lysates; adding purified recombinant GST-MCP to the lysate. My target RNA contains RFP marker and as a control I also transfect similar construct without MS2 tags. I verify the transfection efficiency by RFP signal and generally ~80% of my cells are transfected.
I analysed the results with SDS-PAGE and qPCR, and even though I see the GST-MCP in the elution fraction, I don't detect any significant enrichment of RNA concentration or specifically RFP gene by the qPCR analysis between the control and MS2-tagged samples pulldown. Therefore, I assume that my target mRNA is not binding to GST-MCP or I lose it in a process, could you help me to troubleshoot?
My protocol looks like this:
1. Transfect one 10 cm plate HEK cells with 10 ug of plasmid for each plasmid.
2. Confirm RFP expression next day.
3. Lyse the cells in Lysis Buffer (20 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40, 1mM DTT, EDTA free protease inhibitor, RNAse OUT 0.5 ul/ml).
Buffers I use are matching to this paper: https://doi.org/10.1016/j.ymeth.2012.07.004
4. Clarify lysate by centrifugation.
5. For transfected GST-MCP, mix lysates with RFP-MS2/RFP construct with GST-MCP lysate; for recombinant GST-MCP, add 10 ug of recombinant protein to the lysate
6. Incubate with rotation 4C 3h.
7. Wash glutathione agarose beads (Protino) in the wash buffer (50 mM Tris–HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.05% NP-40) 3x with intermediate centrifugation 1000g, 2 min, 4C.
8. Load similar amount of protein incubated with GST-MCP (I measured by BCA, loaded 1.5 mg) to the washed beads, incubate with rotation 4C 3h.
Besides, I also tried to first immobilize GST-MCP on the glutathione beads and then load the lysate.
9. Centrifuge 1000g, 2 min, 4C, wash with lysis buffer, repeat 3x.
10. Wash 1x in the wash buffer.
11. 10% of the beads I collected for the elution with sample buffer for SDS-PAGE analysis and 90% I used for the RNA extraction (RNA extraction from the similar paper Article Ms2-trap (ms2-tagged RNA affinity purification): Tagging RNA...
)The sequence of the MS2 stem loops I use (24x): tatcgatcctaaggtacctaattgcctagaaa*acatgaggatcacccatgt*ctgcaggtcgactccagaaa