Sample Prep Before a LASSO Pulldown: What Actually Matters

Team Dynamic Matrices | 11 August 2026

When a customer tells us a LASSO pulldown gave low yield or high background, our first question is not about the capture step. It is about the lysate. In most of the cases we have worked through together, the capture chemistry did what it was designed to do on what it was given, and what it was given was the problem. This post is the checklist we run through, in order.

The pulldown is only as good as the sample going in

LASSO captures by hybridisation of catcher strands to the target, followed by a phase transition of the polymer network. Both steps happen in solution and both are sensitive to what else is in that solution. Insoluble debris, unlysed fragments and aggregates get physically entangled in the condensing network and come down with the pellet as background. Chaotropes and high detergent prevent the network from forming. Degraded targets have no intact region for catchers to bind. None of those is a capture failure; all of them look like one.

1. Match the lysis to the target

For RNA targets: gentle detergent lysis (0.5 to 1 percent NP-40 or Triton X-100, physiological salt, RNase inhibitor) preserves structure and keeps the buffer compatible. Chaotropic lysis is fine only if followed by a buffer exchange.

For native protein targets: non-denaturing lysis (NP-40 or digitonin, no SDS, protease inhibitors), on ice. The whole point of native capture is lost if lysis unfolds the protein.

For tissue: you may need mechanical disruption to break it open. Do it cold, briefly, and in a buffer with inhibitors, then proceed as above. Harsh conditions that would be routine for total RNA extraction work against you here.

2. Clear the lysate

Centrifuge at 10,000 to 16,000 g for 5 to 10 minutes at 4 °C and take the supernatant, or pass through a 0.45 µm filter for large volumes. You will lose a small amount of target in the pellet. The reduction in background is worth far more than that loss. Every uncleared lysate we have looked at gave a dirtier pellet.

3. Normalise input on more than concentration

Concentration by fluorometry is necessary and not sufficient. Also check:

• Integrity. RIN or a Bioanalyzer trace for RNA; a Coomassie gel for protein. A degraded RNA sample at the right concentration has few intact catcher-binding regions and will under-capture.
• Target abundance. A quick qPCR for the target (or a western for a protein) before capture tells you whether the target is there at all and lets you calculate capture efficiency afterwards. Without it, a low yield could be a low input.
• Consistency across replicates. If inputs differ in integrity, results will differ, and the difference will be attributed to the capture.

4. Check buffer compatibility

This is the step most often skipped. The polymer phase transition needs approximately physiological ionic strength (100 to 200 mM monovalent salt), neutral pH, detergent under about 1 percent, and no chaotrope or organic solvent carryover. Lysis buffers that are fine for their own purpose can prevent condensation:

• Guanidinium or urea: incompatible; exchange the buffer
• Phenol carryover: incompatible; re-purify
• SDS above 0.1 percent: incompatible for native capture; exchange
• EDTA above 5 mM: reduces hybridisation stability; dilute or exchange
• Glycerol above 10 percent: slows condensation; dilute

A buffer exchange (spin desalting column, or simply dilution into capture buffer if the volume allows) before capture removes the whole class of failure. Build it into the protocol rather than deciding case by case.

5. Scale for low input

For precious samples: scale reaction volumes down proportionally rather than running a small input in a full-size volume, since capture kinetics depend on concentration. Minimise transfers; each one loses a fixed fraction. Use low-binding tubes. Add a carrier if precipitating anything. Consider concentrating dilute samples before capture rather than after.

Pre-capture checklist

• Lysis matched to target (RNA, native protein, tissue)
• Lysate cleared by centrifugation or filtration
• Concentration by fluorometry
• Integrity checked
• Target abundance checked, so efficiency can be calculated
• Buffer at physiological salt, neutral pH, low detergent, no chaotrope
• Volumes scaled to input; transfers minimised

Run through this before the first capture on a new sample type and most troubleshooting never happens.