Polysome profiling protocol step by step SOP with QC and tips

Introduction

Polysome profiling resolves translating ribosomes across sucrose gradients to quantify subunits, monosomes, and polysomes in mammalian cells. This bench-ready SOP walks you through stabilizing translation, preparing 10–50% gradients, ultracentrifugation in SW41Ti, fractionation with UV254 detection, and QC for downstream RNA analysis. To align with search intent, we'll explicitly deliver a polysome profiling protocol for sucrose gradients with reproducible parameters, safety notes, and documentation practices.

Key takeaways

• Hardware: Beckman SW41Ti with 13.2 mL Ultra-Clear (14 × 89 mm) tubes; BioComp Gradient Master for 10–50% linear gradients; ISCO/BioComp fractionation with Teledyne ISCO UA-6 UV254.
• Core buffer: 20 mM HEPES-KOH pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM DTT; 100 μg/mL cycloheximide (CHX) added fresh; RNase/protease inhibitors.
• Spin: 39,000–40,000 rpm, 4 °C, ~2 h in SW41Ti; gentle acceleration, minimal braking.
• Fractionation: 0.5 mL per fraction; baseline-zero UA-6 immediately before the run.
• QC targets: clear 40S/60S/80S separation, polysome:monosome (P:M) ratio > 2 (context-dependent), RIN ≥ 8.0 for sequencing-bound pools, report rRNA proportions.
• Documentation: export A254 traces (CSV/PNG), track rotor/tube lots, buffer recipes, CHX timing, fraction order.

Reagents & setup

Buffers and inhibitors

Polysome buffer (mammalian cells, prepare fresh; keep at 4 °C):

• 20 mM HEPES-KOH, pH 7.4
• 100 mM KCl
• 5 mM MgCl2
• 1 mM DTT (add immediately before use)
• 100 μg/mL cycloheximide (CHX)
• RNase inhibitor (e.g., RNasin, ~40 U/mL)
• Protease inhibitor cocktail

Sucrose solutions (filter-sterilized, pre-chilled 4 °C):

• 10% (w/v) sucrose in polysome buffer
• 50% (w/v) sucrose in polysome buffer

DrummondLab offers practical gradient tips for preparing 10% and 50% sucrose in the correct buffer system; see the succinct guidance in the sucrose gradient protocol page: DrummondLab: Sucrose gradient protocol.

Equipment and rotors

• Beckman Coulter SW 41 Ti swinging-bucket rotor; official specifications list max 41,000 rpm, max 288,000 × g, k-factor 124, compatible with 14 × 89 mm tubes: Beckman SW 41 Ti rotor specifications.
• 13.2 mL Ultra-Clear open-top thinwall tubes, 14 × 89 mm (compatible with SW41Ti).
• BioComp Gradient Master (Model 108/153) for casting linear 10–50% gradients in 14 × 89 mm tubes (consult your device manual for program specifics; see vendor support index: BioComp Gradient Master manuals).
• BioComp/ISCO fractionation with Teledyne ISCO UA-6 UV/VIS detector for 254 nm monitoring (UA-6 baseline zeroing and attenuation workflows: UA-6 Operator Manual, Rev. C).

RNase-free practices

• Use RNase-free plastics and filtered tips; dedicate reagents and ice buckets for RNA work.
• Keep all buffers, tubes, and samples at 4 °C; work on ice immediately after CHX addition.
• Add RNase inhibitors to lysis and gradient buffers; prepare DTT fresh; avoid repeated freeze–thaw.
• Wipe work surfaces with RNase decontamination solution; change gloves frequently.

Cell preparation & lysis

Cycloheximide pre-treatment

• Add CHX to culture medium at 100 μg/mL for 5–10 minutes prior to harvest; maintain arrest by including CHX in wash and lysis buffers. Multiple mammalian workflows use this timing and concentration; examples report 0.1 mg/mL CHX for 5–10 min before lysis alongside 10–50% gradients and SW41Ti spins at 4 °C and ~2 h, as shown in peer-reviewed studies (e.g., 2022–2024 reports in PMC cohorts).
• Caveat: CHX can introduce artifacts in some contexts; match to your biological question and include appropriate controls.

Lysis buffer and steps

1. Chill all buffers and consumables; pre-cool rotor buckets.
2. Aspirate medium; rinse once with ice-cold PBS containing 100 μg/mL CHX.
3. Add ice-cold lysis buffer (polysome buffer + CHX + RNase/protease inhibitors). Use volumes appropriate to cell pellet or dish area (e.g., ~0.5–1.0 mL per 10 cm dish).
4. Perform gentle mechanical lysis (pipetting up/down; avoid harsh detergents). Keep on ice.
5. Incubate 5 minutes on ice; briefly clear viscous debris by passing through a 26G needle if needed.

Clarification and quantification

• Clarify lysate at 12,000 × g for 10 minutes at 4 °C; retain supernatant.
• Quantify nucleic acids by A260 (Nanodrop or plate reader). Normalize inputs per gradient (e.g., 10–50 μg RNA equivalent), noting experiment goals.

Sucrose gradient preparation

Gradient recipes and ranges

• Prepare 10% and 50% sucrose in polysome buffer; keep both at 4 °C.
• For 13.2 mL Ultra-Clear tubes (14 × 89 mm), aim for ~11–12 mL total gradient volume, leaving headspace for sample overlay.

Linearization methods

• BioComp Gradient Master: load 10% and 50% solutions per device instructions to generate linear 10–50% gradients in 14 × 89 mm tubes. Program parameters (angle, speed, time) are tube- and model-specific; consult the manual or vendor support for the correct program and tube calibration steps: BioComp Gradient Master manuals.
• If a Gradient Master is unavailable, a manual gradient maker can be used; keep tubes upright and minimize disturbance. Practical formation tips are described in DrummondLab's protocol.

Tube prep and balancing

• Label tubes; mark meniscus heights for consistent fills.
• Seal tubes appropriately; avoid air bubbles.
• Precisely balance tube pairs by mass (≤ 0.1 g difference); pre-chill tubes and buckets at 4 °C.

Ultracentrifugation & fractionation

Spin parameters and handling

• Load clarified lysate gently onto gradients (top overlay). Fill volumes should not exceed tube limits.
• Spin in SW41Ti at 39,000–40,000 rpm, 4 °C, ~2 hours. Typical mammalian protocols report 40,000 rpm for ~2 h at 4 °C in SW41Ti-class rotors, enabling resolution of 40S/60S/80S and polysomes; verify RPM↔RCF on your instrument. The SW41Ti's official limits (max 41,000 rpm; 288,000 × g; k-factor 124) are published by Beckman Coulter: SW41Ti rotor specs.
• Use gentle acceleration; disable brake or choose low deceleration to preserve gradient shape. Handle tubes without bending; avoid bucket drag.

Fractionation and UV254 profiling

• Integrate the gradient outlet with the BioComp/ISCO fractionation system routed through the UA-6 at 254 nm.
• Set fraction size to 0.5 mL; expect ~22–24 fractions from an ~11–12 mL gradient (plus overlay).
• UA-6 setup: zero the baseline immediately before fractionation (ZERO control), choose appropriate attenuation/AUFS to prevent clipping, and verify analog output range for recorder/fraction collector triggering as described in the UA-6 manual: UA-6 Operator Manual.
• Expected profile: discrete 40S, 60S, 80S peaks followed by progressively larger polysomes toward the bottom. Recent analyses in mammalian systems explain how shifts in 80S vs polysome peaks relate to initiation and elongation changes; see interpretive guidance in MBoC 2025 on polysome profiles.

Representative sucrose gradient polysome profile and fractionation scheme. The diagram illustrates expected UV254 trace peaks for 40S, 60S, 80S monosomes, and polysomes following ultracentrifugation, aligned with sedimentation position and collection tubes. A Polysome-to-Monosome (P:M) ratio > 2 is highlighted as a key quality control target for actively translating cells.

Data capture and storage

• Export A254 traces as CSV and PNG; save fraction IDs and volumes.
• Record metadata: ultracentrifuge and rotor IDs, tube lot numbers, buffer recipes/lot numbers, CHX timing/concentration, temperature, fractionation flow rate and AUFS, operator, date, deviations.
• Use consistent file naming (e.g., YYYYMMDD_CellLine_SW41Ti_10-50pct_UA6.csv).

QC & troubleshooting

QC metrics and targets

• UV254 trace quality: clear separation of 40S/60S/80S peaks; low baseline noise; no clipping.
• Polysome:monosome ratio (P:M): target > 2.0 for healthy, actively translating cultures; interpret relative to biological condition (stress may lower P:M) and reported controls.
• RNA integrity for sequencing-bound fractions/pools: RIN ≥ 8.0 (strict threshold improves downstream library prep success). Many workflows accept RIN ≥ 7; we apply ≥ 8.0 here for stricter acceptance.
• Purity benchmarks: OD260/280 ≈ 1.8–2.2; OD260/230 ≥ 2.0; DV200 ≥ 70% acceptable if RIN is marginal. See sample prep QC guidance for definitions and acceptance ranges in an educational overview: RNA quality metrics: RIN/DV200, OD ratios.
• rRNA proportion reporting: quantify rRNA content in each fraction or pooled set; for mRNA-focused sequencing, aim to reduce rRNA proportion in polysome-enriched pools. Educational context on strategy selection is summarized here: Overview of rRNA depletion strategies.

Common issues and fixes

• Collapsed polysomes (weak polysome peaks, inflated 80S)
  • Causes: insufficient CHX timing/concentration; delayed lysis; low Mg2+; RNase activity; cellular stress.
  • Fixes: verify CHX (100 μg/mL for 5–10 min); maintain cold chain; ensure MgCl2 at 5 mM; add RNase inhibitors; include an EDTA-treated control to confirm peak identities. Stress-induced collapse patterns are shown in 2024 cohorts: stress-induced polysome collapse.
• Noisy or broad UV baseline
  • Causes: bubbles, dirty flow cell/tubing, incorrect attenuation.
  • Fixes: degas buffers; clean or replace the UA-6 flow cell; adjust AUFS; re-zero baseline immediately before fractionation (see UA-6 manual).
• Poor peak resolution
  • Causes: gradient non-linearity; overfilled tubes; rough handling; high acceleration/deceleration.
  • Fixes: verify Gradient Master program and tube calibration; reduce acceleration/braking; adhere to ~11–12 mL fills; keep gradients strictly at 4 °C. Practical formation notes: DrummondLab gradient protocol.
• Low RNA yield in polysome fractions
  • Causes: inefficient extraction; sucrose interference; cleanup losses.
  • Fixes: pool adjacent fractions to reach input targets; add glycogen carrier; prefer column-based cleanup; consider RAPPL for sucrose removal prior to extraction: RAPPL method for sucrose removal (NAR 2024).

Downstream RNA readiness

• Sucrose removal and cleanup: remove sucrose before extraction. RAPPL (poly-lysine magnetic bead affinity) efficiently removes sucrose while preserving ribosomes: NAR 2024 RAPPL. Alternatives include benchtop pelleting to discard supernatant or sucrose cushion pelleting; direct extraction is possible but may carry contaminants.
• Minimal inputs for common kits: consult your library kit's low-input specifications; pool fractions if needed and apply DNase treatment post-cleanup.
• Neutral educational brand context and disclosure: Disclosure: CD Genomics is our product. CD Genomics can support fraction RNA QC (RIN/DV200 assessment), advise on rRNA depletion strategy selection (poly(A) enrichment vs RNase H-based or Ribo‑Zero‑like depletion), and provide low‑input library preparation support for polysome‑derived RNA. For criteria and method selection, see the educational overviews on RNA quality metrics and rRNA depletion strategies. For workflow context, a ribosome profiling overview is available: ribosome profiling service context.

Conclusion

With this protocol, you should resolve 40S, 60S, 80S (monosome), and polysomes with defined QC targets (P:M ratio, RIN, rRNA proportion) and documented parameters. Proceed to fraction-resolved analyses such as RT-qPCR or RNA-seq according to your study design. Maintain complete records—rotor and tube IDs, buffer lots, CHX timing, UA-6 settings, and trace exports—to ensure reproducibility across replicates.

FAQ

• • What is the recommended cycloheximide (CHX) concentration and timing to preserve polysomes?

    • Use 100 μg/mL CHX (0.1 mg/mL) added to culture medium 5–10 minutes before harvest and maintain CHX in wash and lysis buffers to preserve arrested ribosomes; validate with a small control because CHX can introduce context-dependent artifacts.

• • How should I pool fractions for sequencing to maximize mRNA content while minimizing rRNA?

    • Pool contiguous polysome-enriched fractions guided by the UV254 trace (exclude heavy rRNA-rich peaks if present), quantify RNA per pool, and aim for pools with reduced rRNA proportion; if necessary, apply rRNA-depletion rather than poly(A) selection for non-polyadenylated targets.

• • My fraction RNA input is low—what library prep approach is recommended?

    • For low-input polysome RNA, pool fractions to reach kit minimums, use low-input or amplification-compatible library kits, and consider rRNA-depletion workflows suited to small inputs; include a carrier during cleanup to maximize recovery.

• • The UA-6 baseline drifts or is noisy—what quick checks should I run?

    • Degas and filter buffers, flush and inspect the flow cell/tubing for bubbles or deposits, verify UA-6 attenuation/AUFS settings and re-zero baseline immediately before the run; replace or clean the flow cell if noise persists.

• • Why use 0.5 mL fractions rather than larger or smaller volumes?

    • 0.5 mL balances resolution and practicability for 11–12 mL 13.2 mL gradients—smaller fractions increase resolution but raise handling and cleanup burden, while larger fractions reduce peak discrimination and may dilute signal for downstream assays.

• • I observe polysome collapse (strong 80S, weak polysomes)—what are the first troubleshooting steps?

    • Confirm CHX timing and presence in all buffers, maintain cold chain and add RNase inhibitors, check Mg2+ concentration (typically 5 mM), run an EDTA-treated control to verify peak identities, and review lysis harshness and handling delays as likely causes.