Hi everyone, I am performing immunoprecipitation (IP) of Flag-Ubl3 protein using anti-Flag tag antibody magnetic beads. During the IP, I need to perform a ‘harsh wash’ so that only proteins that are UBL3-modified remain. Which conditions of the washing buffer do I need to consider for `harsh washing`?
For harsh washing conditions in washing buffers, you should consider using high salt concentrations (like 0.5–1 M NaCl), detergents (e.g., SDS, Triton X-100), and chelators (such as EDTA) to disrupt non-specific interactions. Adjusting the pH to be more acidic (pH 3–5) or basic (pH 9–11) can also help, as well as increasing the temperature (37–65°C) to weaken bonds. Organic solvents like ethanol or methanol can be added to remove hydrophobic interactions. Ensure that the conditions are suitable for your specific target, as harsh conditions can damage sensitive molecules
Junaid Nazir Thank you for your response. Do you have the exact formulation of the washing buffer?
Harsh Washing Buffer Formulation:
Tris-HCl: 10–50 mM (pH 7.5–9.0) – Maintains buffering capacity.
NaCl or KCl: 0.5–1 M – Disrupts weak ionic interactions.
SDS: 0.1–1% – Disrupts hydrophobic interactions.
EDTA: 1–10 mM – Chelates metal ions, preventing metal-dependent interactions.
Triton X-100 or Tween-20: 0.1–2% – Non-ionic detergents for mild disruption of protein-protein interactions.
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