Frequently Asked Questions

for OneMinute® Western Blot Stripping Buffers

Questions

  1. Does OneMinute® stripping buffer work for Amersham ECL, ECL Plus and ECL Advance?
  2. How to know whether the stripping removes the primary and secondary antibodies completely?
  3. How to optimize the stripping conditions for best result?
  4. Can OneMinute® stripping buffer be used on both nitrocellulose and PVDF membranes?
  5. Do I need to re-block the membrane after stripping?
  6. Will OneMinute® stripping buffer remove chemiluminescent substrate precipitated on the membrane?
  7. How many times can I strip and re-probe the same membrane using OneMinute®?
  8. In what scenarios, OneMinute® does not work very well?
  9. Does OneMinute® stripping buffer expire?
  10. Can OneMinute® quench/remove fluorescent signals generated from chemifluorescence?
  11. I used fluorescent dyes conjugated secondary antibody in my Western Blot. Does OneMinute® work in this scenario?
  12. Is there a simple rule that I can follow to use OneMinute® successfully?

Answers

1. Does OneMinute® stripping buffer work for Amersham ECL, ECL Plus and ECL Advance?

Yes.

  • OneMinute® works very well for ECL and ECL Advance.
  • OneMinute® works very well for ECL Plus if your membrane is exposed to X-ray film, not a scanner or CCD camera.
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2. How to know whether the stripping removes the primary and secondary antibodies completely?

  • Test for complete removal of secondary antibodies: After stripping, incubate the membrane with fresh chemiluminescent substrate (such as ECL) and expose to X-ray film. If no signal is detected after a 5-minute exposure, the secondary antibodies have been successfully removed.
  • Test for complete removal of the primary antibodies: Incubate the membrane with HRP-conjugated secondary antibodies, followed by 3 washes. Incubate with fresh substrate and expose to X-ray film. If no signal is detected after a 5-minute exposure, the primary antibodies have been successfully removed.
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3. How to optimize the stripping conditions for best result?

Generally, OneMinute® removes antibodies completely in 30 sec to 1 min. If antibodies are not completely removed, try to:

  • Increase stripping time up to 5 minutes while shaking.
  • Increase incubation temperature to 37°C. However, this may reduce the signal of the target protein.
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4. Can OneMinute® stripping buffer be used on both nitrocellulose and PVDF membranes?

Yes. OneMinute® stripping buffer efficiently removes antibodies from both nitrocellulose and PVDF membranes.

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5. Do I need to re-block the membrane after stripping?

Re-blocking the membrane is usually not necessary after stripping with OneMinute®, but it is recommended to re-probe the membrane with antibodies in a buffer containing 1% milk. However, re-blocking may help to decrease background in some situations.

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6. Will OneMinute® stripping buffer remove chemiluminescent substrate precipitated on the membrane?

No, but the luminescent signals are quenched. HRP or AP conjugated to the secondary antibody cleaves a chemiluminescent substrate, and the reaction product produces a luminescent signal and precipitates on the membrane. OneMinute® strips off primary/secondary antibodies and quenches the luminescent signals, but it cannot remove the precipitate.

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7. How many times can I strip and re-probe the same membrane using OneMinute®?

OneMinute® Stripping buffer is harsh on primary and secondary antibodies but very gentle on antigens bound to the membrane. In one of our tests, 50% of the antigen still remained on the membrane after 10 stripping cycles (see Figure 6). However, results may vary depending on the stability of the antigen and the sensitivity of the antibodies. Antigens may withstand as many as 10 stripping cycles or as few as one.

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8. In what scenarios, OneMinute® does not work very well?

  • OneMinute® cannot break strong interactions, such as the avidin-biotin interaction.
  • In some rare cases, OneMinute® may not work well for specific high-affinity antibodies.
  • OneMinute® does not strip off fluorescent signals.
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9. Does OneMinute® and OneMinute® Plus stripping buffer expire?

Yes. OneMinute® Stripping buffer expires in several months. OneMinute® Plus has a much longer shelf life (~18 months). Please find the expiration date on the bottle.

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10. Can OneMinute® quench/remove fluorescent signals generated from chemifluorescence?

No. In the chemifluorescent method, non-fluorescent substrates are enzymatically converted into fluorescent products by HRP or AP conjugated to the secondary antibody. The fluorescent products precipitate on the membrane. OneMinute® strips off primary and secondary antibodies but cannot remove or quench the fluorescent precipitates.

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11. I used fluorescent dye-conjugated secondary antibodies in my Western Blot. Does OneMinute® work in this scenario?

No. Unacceptable fluorescent background could be generated although the antibodies can be stripped off. OneMinute® is designed for procedures using chemiluminescent substrates.

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12. Is there a simple rule that I can follow to use OneMinute® successfully?

Yes. Keep in mind to expose your membrane to X-ray film for signal detection. OneMinute® works very well in procedures where X-ray film is used to detect signals in subsequent re-blotting.

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