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Factors that Affect Stripping Efficiency

Certain factors can affect the efficiency of any stripping buffer, depending on the type of stripping buffer and membrane used.

When Using NewBlot IR Stripping Buffer

Recommended Standard Conditions: 1X NewBlot IR for 20 minutes at room temperature.

Below are key factors that affect stripping efficiency with NewBlot IR on Odyssey™ nitrocellulose or Immobilon®-FL PVDF membranes.

  • Amount of time blot is in stripping buffer
    • Increasing the stripping time may increase stripping efficiency, however, it may also remove target proteins and reduce the success of reprobing.
  • Sample type and preparation
    • Even under the most stringent stripping conditions, the fluorescent signal may not be removed completely due to sample load amount, antibody affinity/avidity, and target protein abundance.


      NewBlot IR Stripping Buffer is not recommended for loading amounts over 30 µg.

  • Blot handling conditions
    • Washing, scanning, or stripping efficiency will be affected if the blot is allowed to dry at all during incubation. Keep the blot moist at all times.
  • Buffer concentration and temperature used for stripping
    • Increasing the stripping buffer concentration or temperature will reduce stripping efficiency, and is not recommended for NewBlot IR Stripping Buffer. Use the material at a 1X concentration at room temperature for optimal results.

When Using NewBlot Nitro Stripping Buffer

Recommended Standard Conditions: 1X NewBlot Nitro, 5 minutes at room temperature.

Below are key factors that affect stripping efficiency with NewBlot Nitro on Odyssey nitrocellulose membranes. Figure 1 compares stripping efficiency using various concentrations, time, and temperature to the recommended standard conditions, 1X, 5 min, ambient.

  • Amount of time blot is in stripping buffer
    • Increasing stripping time has the greatest effect on efficiency.
    • Increasing the stripping time may lead to increased damage/loss of target antigens, and reduce the success of reprobing.
  • Buffer concentration and temperature used for stripping
    • Increasing the stripping buffer concentration and temperature significantly improves stripping effectiveness, but can also have a highly detrimental effect on reprobing.
    • If the optimization process given in the protocol does not produce the desired stripping results, Stripping Buffer incubation can be carried out at 37 °C using a water bath or warm-air incubator.
  • Temperature used for stripping
    • Increased temperature may improve effectiveness, but can be highly detrimental for reprobing.
    • If the optimization process given in the protocol does not produce the desired stripping results, Stripping Buffer incubation can be carried out at 37 °C using a water bath or warm-air incubator.
    • Do not microwave the NewBlot Nitro Stripping Buffer or the nitrocellulose blot.
  • Sample type and preparation
    • Even under the most stringent stripping conditions, the fluorescent signal may not be removed completely due to sample load amount, antibody affinity/avidity, and target protein abundance.
  • Blot handling conditions
    • Washing, scanning, or stripping efficiency will be affected if the blot is allowed to dry at all during incubation. Keep the blot moist at all times.

When Using NewBlot PVDF Stripping Buffer

Recommended standard conditions:  1 X NewBlot PVDF for 20 min at room temperature.
Incubation time, buffer concentration, and temperature affect stripping efficiency

Figure 2. Images showing an example of Western blot stripping optimization with NewBlot PVDF Stripping Buffer. (A) Initial Western blot, showing EGFR detected with IRDye 800CW goat anti-mouse and β−Actin detected with IRDye 680LT goat anti-rabbit. (B) After stripping with standard stripping Procedure. (C) After stripping for an additional 20 min. (D) After additional 5 min stripping with the addition of SDS. (E) After 20 minutes total stripping time in NewBlot PVDF + SDS. (F) Reprobe image showing ERK2 detected with IRDye 680LT goat anti-mouse and β−Tubulin detected with IRDye 800CW goat anti-rabbit.

Below are factors that affect stripping efficiency with NewBlot PVDF on PVDF membranes. Figure 1 shows the effect of incubation time and addition of SDS on stripping efficiency. These blots and protein targets were not sufficiently stripped using the standard Procedure, and optimization was performed.

For optimal stripping results, follow the optimization guidelines in NewBlot PVDF Stripping Buffer Protocol.

  • Amount of time blot is in stripping buffer
    • Stripping time has the greatest effect on efficiency.
    • Increasing the stripping time may lead to increased damage/loss of target antigens, and reduce the success of reprobing.
  • Blot handling conditions
    • Washing, scanning, or stripping efficiency will be affected if the blot is allowed to dry at all during incubation. Keep the blot moist at all times.
  • Detergent may help remove fluorescent signal
    • If fluorescent signal remains, try longer incubation times first, followed by addition of SDS.
  • Temperature used for stripping
    • If fluorescent signal remains after the above steps, incubation in stripping buffer may be carried out at 37 °C using a water bath or incubator.  This should only be attempted if the above optimization steps are unsuccessful.
  • Sample type and preparation
    • Even under the most stringent stripping conditions, the fluorescent signal may not be removed completely due to sample load amount, antibody affinity/avidity, and target protein abundance.



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