Stripping & Reprobing Blots

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It is possible to strip the detection antibodies off of a western blot, and then re-probe that blot with a second set of antibodies.  Every strip will reduce the amount of available protein, so lower-abundance proteins should be probed for prior to higher-abundance proteins.  Two methods to strip blots are commonly used:

Contents

Acid Stripping

Acid stripping relies on a low-pH buffer to interrupt the ionic bonds through which antibodies tend to bind their ligands.  Most antibodies can be removed by washing at room temp with low (pH 2.0 to 2.5) buffer.  Importantly, this procedure does not denature many proteins, making it ideal for detecting non-denatured epitopes.  However, it is less robust than other striping methods, and as such may not remove all antibodies.

Stripping Buffer

2.5M glycine, 1% SDS in water, pH 2.0 to 2.5.

  • 1.876g glycine
  • 50ml of 20% SDS
  • 950ml ddH2O

Dissolve glycine in water, then add SDS.  pH to 2.5 or lower using 6M or conc. HCl.

Stripping Procedure
  1. Wash blot 1x 5min in TBST to remove any ECL or other residues
  2. Wash 3 x 10min in stripping buffer at room temperature
  3. Wash blot 1 x 5min in TBST to restore pH
  4. Re-block probe in TBST + desired blocking agent for 1 hour at room temperature
  5. Probe with primary/secondary antibodies as per normal procedures.

Denaturation Striping

Denaturation striping relies on heat and a reducing agent (β-mercaptoethanol) to denature the antibodies.  While this will remove even the most stubborn of antibodies, it also denatures any proteins on the blot, and tends to greatly reduce the amount of protein remaining on the membrane.

Denaturation Buffer

50mM tris pH6.8, 100mM β-mercaptoethanol and 2% SDS

  • 605mg Tris
  • 700ul β-mercaptoethanol
  • 10ml of 20% SDS

Dissolve Tris and SDS in water; pH to 6.8, then add β-mercaptoethanol.

Stripping Procedure
  1. Wash blot 1x 5min in TBST to remove any ECL or other residues
  2. Seal blot with 100ml of denaturation buffer in a container, and immerse in 50C water bath for 30min.  Shake, if possible.
  3. Wash blot 1 x 5min in TBST to restore pH
  4. Re-block probe in TBST + desired blocking agent for 1 hour at room temperature
  5. Probe with primary/secondary antibodies as per normal procedures.
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