Size-Exclusion Chromatography of Argonaute-Containing Complexes

Size-Exclusion Chromatography for the Analysis of Argonaute-Containing Ribonucleoprotein Complexes in Mammalian Cells and Tissues

Introduction

This protocol describes size-exclusion chromatography (SEC) to separate Argonaute (AGO)-containing ribonucleoprotein complexes from mammalian cells and tissues according to apparent molecular size. Clarified native extracts are fractionated on a Superose 6 column; proteins from individual fractions are recovered by trichloroacetic acid (TCA) precipitation and analyzed by Western blotting. The method allows comparison of AGO distributions between high- and low-molecular-weight regions (HMWR and LMWR).

Materials and reagents

Reagents

• NaCl (5 M stock); Tris-HCl, pH 7.5 (1 M stock); MgCl₂ (1 M stock)
• Triton X-100 (10% stock); DTT; protease and phosphatase inhibitor cocktails
• RNase inhibitor (optional); DEPC-treated water; PBS
• TCA (100%); acetone; LDS sample buffer (4×); reducing agent (10×)
• Anti-AGO2 antibody: Cell Signaling Technology, clone C34C6, cat. no. 2897 (WB; published dilution 1:1,000)
• SDS-PAGE and Western blotting reagents, including nitrocellulose membrane and appropriate secondary antibody

Equipment

• FPLC system with injection valve and fraction collector
• Superose 6 10/300 GL (GE Healthcare, cat. no. 17-5172-01; discontinued); replacement: Superose 6 Increase 10/300 GL (Cytiva, cat. no. 29-0915-96). See Column notes.
• Dounce homogenizer; precooled mortar and pestle for frozen tissues
• Refrigerated microcentrifuge; vortex mixer; heating block
• SDS-PAGE and Western blotting equipment

Buffer preparation

SUP6-150 buffer (1 L)

Immediately before use, supplement the required buffer volume with 1 mM DTT, protease inhibitors, phosphatase inhibitors, and optionally RNase inhibitor. In our laboratory experience, RNase inhibitors are not strictly required for most tissue samples.

Procedure

A. Sample collection and preparation

1. Harvest tissues or collect cultured cell pellets. Use enough starting material to recover up to 2 mg total soluble protein for each SEC run; determine the required input empirically.
2. When appropriate, perfuse mice with cold PBS before tissue collection to reduce blood-derived contamination.
3. Snap-freeze tissues or cell pellets in liquid nitrogen and store at −80°C until use. Given the low detergent concentration (0.01% Triton X-100), freezing facilitates subsequent mechanical disruption; avoid repeated freeze–thaw cycles.
4. If necessary, pulverize frozen tissue with a precooled mortar and pestle. Keep samples cold during preparation.

B. Preparation of native lysates

1. Prepare 500 µL of supplemented SUP6-150 buffer per sample. Target no more than 2 mg soluble protein in up to 500 µL clarified lysate for injection.
2. For tissue, transfer the sample into a Dounce homogenizer containing 500 µL supplemented buffer. For cultured cell pellets, resuspend directly in the buffer without Dounce homogenization.
3. For tissue, homogenize gently using five Dounce strokes followed by 10 seconds on ice; repeat ten times.
4. Avoid excessive force and sample heating. Transfer homogenate to a microcentrifuge tube.
5. Clarify by centrifugation for 5 min at 4°C.
6. Transfer the supernatant without disturbing debris or the surface lipid layer. Repeat clarification as needed, then keep lysate on ice.
7. Measure soluble protein concentration using a compatible assay and calculate the injection volume.

C. Size-exclusion chromatography

1. Connect the Superose 6 10/300 GL column to an FPLC system. Use degassed SUP6-150 buffer as the mobile phase and observe the manufacturer’s column-pressure limit.
2. Equilibrate with SUP6-150 buffer until the baseline is stable.
3. Set the separation flow rate to 0.3 mL/min.
4. Inject up to 500 µL clarified extract containing no more than 2 mg soluble protein; avoid introducing bubbles. For direct reproduction of La Rocca et al. (2021), use 400 µL containing 1.5–2 mg protein.
5. Elute with SUP6-150 buffer and collect consecutive 1 mL fractions, recording the elution volume corresponding to each fraction.
6. Keep collected fractions on ice and proceed promptly to TCA precipitation; if storage is necessary, freeze at −80°C and avoid repeated freeze–thaw cycles.

D. TCA precipitation of SEC fractions

1. Transfer each 1 mL SEC fraction to a labeled, TCA-compatible tube.
2. Add 300 µL of 100% TCA per fraction and invert five times to mix.
3. Incubate at −80°C overnight.
4. Centrifuge for 15 min at 16,100 × g; carefully remove supernatant without disturbing the precipitate.
5. Wash with 500 µL cold acetone; centrifuge for 5 min at 4°C.
6. Remove acetone carefully. Precipitated proteins may appear as a thin opaque film rather than a discrete pellet.

E. Preparation for SDS-PAGE

1. Prepare fresh 1× LDS loading buffer containing 1× reducing agent, diluted in PBS or another compatible diluent.
2. Add 30 µL loading buffer to each precipitated sample.
3. Vortex briefly (~3 s), heat at 60°C for 1 min, then vortex for ~10 s.
4. Heat at boiling temperature for 8 min, vortexing approximately every 2 min; briefly centrifuge.
5. Load onto 4–12% Bis-Tris SDS-PAGE gels, separate proteins, and transfer to nitrocellulose membrane.

F. Western blot analysis

1. Block the membrane with an appropriate blocking reagent (e.g., 5% nonfat dry milk).
2. Incubate with anti-AGO2 (CST #2897; 1:1,000 as reported in La Rocca et al., 2021), or another validated antibody.
3. Wash, incubate with the appropriate secondary antibody, and detect by chemiluminescence or another validated method.
4. Compare AGO2 signals across consecutive fractions. Optionally probe for other RISC-associated proteins, including TNRC6 family members.

Data analysis

Larger assemblies generally elute earlier than smaller assemblies. Define HMWR and LMWR fraction ranges from column calibration and observed elution profiles, not from a universal fraction-number cutoff. For each lane, quantify background-corrected AGO2 band intensity (Iᵢ); relative abundance in fraction i (%) = 100 × Iᵢ / ΣIⱼ. Plot abundance against fraction number or elution volume, and sum normalized intensities within calibrated HMWR and LMWR windows. SEC reflects hydrodynamic behavior; apparent molecular masses should be interpreted cautiously.

Notes and troubleshooting

Preservation and sample handling

Maintain cold handling and gentle lysis. Clarify particulate material thoroughly without discarding soluble complexes. Use consistent input, buffer composition, sample volume, fraction collection, and detection conditions across samples.

Column availability and alternatives

The original Superose 6 10/300 GL column (GE Healthcare, cat. no. 17-5172-01) has been superseded by Superose 6 Increase 10/300 GL (Cytiva, cat. no. 29-0915-96). Both have 10 × 300 mm dimensions and approximately 24 mL bed volume, but the Increase resin has smaller particles (~8.6 µm versus ~13 µm), with potentially different resolution and AGO elution profiles. In our laboratory experience, the original column exhibited a longer useful operating lifespan, although this observation was not established by a controlled longevity comparison. Superose 6 prep grade bulk resin (cat. nos. 17-0489-01 and 17-0489-03; ordering availability to be verified) can be considered for custom packing; its ~30 µm particles are not equivalent to the analytical resin. Alternative columns must be calibrated and the HMWR/LMWR boundaries re-established.

Expected results

AGO proteins can distribute between HMWR and LMWR fractions depending on sample type and experimental conditions. Disruption of AGO–TNRC6 interactions by T6B can shift AGO toward LMWR fractions, as described in the cited studies.

References

La Rocca G et al. (2015). In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA. Proc Natl Acad Sci USA 112:767–772. doi:10.1073/pnas.1424217112.

La Rocca G et al. (2021). Inducible and reversible inhibition of miRNA-mediated gene repression in vivo. eLife 10:e70948. doi:10.7554/eLife.70948.

Cytiva. Superose 6 10/300 GL and Superose 6 Increase 10/300 GL: manufacturer instructions and size-exclusion chromatography documentation.