Isolation of Small Regulatory RNA–Bound Bacterial Target RNA Using Affinity Purification

Samenvatting

Source: Mercier, N., et. al., MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)

This video demonstrates the isolation of MS2-tagged small regulatory RNA (sRNA) bound to its bacterial target RNA using affinity purification with a maltose-binding protein (MBP)–MS2 coat protein fusion immobilized on amylose resin.

Protocol

Buffers and media

For MAPS experiments, prepare the following buffers and media:- Buffer A (150 mM KCl, 20 mM Tris-HCl pH 8, 1 mM MgCl₂ and 1 mM DTT)- Buffer E (250 mM KCl, 20 mM Tris-HCl pH 8, 12 mM maltose, 0.1% Triton, 1 mM MgCl₂ and 1 mM DTT)

Column preparation

CAUTION: Be careful not to allow the amylose resin to dry. If needed, seal the column with an end-cap. Prepare all the solutions before starting the affinity purification.

1. Put a chromatography column in a column rack.
2. Remove the column tip and wash the column with ultrapure water.
3. Add 300 µL of amylose resin.
4. Wash the column with 10 mL of Buffer A.
5. Dilute 1,200 pmol of MBP (Maltose-binding protein)-MS2 protein in 6 mL of Buffer A and load it into the column.
6. Wash the column with 10 mL of Buffer A.

MS2-affinity purification (Figure 1)

1. Load the cell lysate into the column.
   NOTE: Keep 1 mL of the cell lysate (Crude extract, CE) to extract total RNA and perform Northern blot and transcriptomic analysis.
2. Collect the flow-through fraction (FT) in a clean collection tube.
3. Wash the column 3 times with 10 mL of Buffer A. Collect the wash fraction (W).
4. Elute the column with 1 mL of Buffer E and collect the elution fraction (E) in a 2 mL microtube.
5. Keep all collected fractions on ice until RNA extraction or freeze them at -20 °C for later use.

Resultaten

Figure 1. Schematic illustration of the MAPS protocol adapted to Staphylococcus aureu...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
1.5 mL microcentrifuge tube	Sarstedt	72.690.001
15 mL centrifuge tubes	Falcon	352070
2 mL microcentrifuge tube	Starstedt	72.691
250 mL culture flask	Dominique Dutscher	2515074	Bacterial cultures
50 mL centrifuge tubes	Falcon	352051	Culture centrifugation
Amylose resin	New England BioLabs	E8021S	MS2-affinity purification
pMBP-MS2	Addgene	65104	MS2-MBP production
Poly-Prep chromatography column	BioRad	7311550	MS2-affinity purification