NewBlot IR Stripping Buffer is supplied as a 5X concentrated solution. Each 100 mL bottle is sufficient for up to 3000 cm2 or approximately fifty 7 × 8.5 cm Odyssey® nitrocellulose membranes.
Factors Affecting Efficiency 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.
For optimal stripping results, follow the optimization guidelines in NewBlot IR Stripping Buffer pack insert.
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.
Note: 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.