Stripping of Digoxigenin-labeled Probes from Nylon Membranes

Reference: Dubitsky, A, and DeFiglia, JA, BioTechniques 1995: 19: 210-212.

Introduction

Moller, Schilling and Karlovsky(3) were unable to strip digoxigenin (DIG)-labeled probes from nylon membranes. They pointed out, rightly, that in the case of DIG-labeled probe, the combination of proteinase K and formamide effectively removed or deactivated conjugate but did not dissociate the underlying probe target hybrid DNA. New data from our laboratory confirm these results but also show that probe can be stripped from the membranes using a solution containing 0.2M NaOH and 0.1% sodium dodecyl sulfate (SDS).

In our original experiments(2), we added fresh substrate after stripping to determine whether any residual signal could be generated. Lack of signal was considered to be equivalent to effective stripping. This was valid for the Amersham ECL™ system, which was tested side by side with the Boehringer Mannheim Genius™ system in our article. The ECL system uses a probe labeled directly with peroxidase. If the peroxidase was deactivated but the probe remained on the membrane, we would not be able to successfully reprobe the original sites. Successful reprobes demonstrated that the probe was removed with the enzyme.

Recent additions to the Boehringer Mannheim product literature(1) include an alkali stripping method. We re-evaluated our stripping procedures per Boehringer Mannheim's guidelines and incorporated modifications suggested in Moller's article. Results showed that probe was removed from the membrane and that the signal could be re-generated five times (4 re-probes).

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Materials and Methods

Thirty nanograms of lamdba HindIII DNA were added to all lanes on 1% agarose gels. After electrophoresis, the DNA was transferred to Biodyne® Plus positively charged nylon membranes (Pall Corporation, Port Washington, NY, USA). Membranes were fixed by UV exposure and hybridized with 0.8-80 ng/mL of DIG-labeled lambda probe (alkali stable, prepared using the Genius™ 2 Labeling Kit [Roche Applied Science, Indianapolis, IN USA]). Membranes were washed, incubated with conjugate (Genius 1 Kit; Roche Applied Science) and developed with Lumi-Phos™ 530 substrate (Lumigen, Southfield, NJ, USA).

After development, membranes were stripped by (i) incubation in 0.2 M NaOH, 0.1% SDS for 30 min at 37 °C or by (ii) pretreatment with protein ase K, 0.5 mg/mL (Sigma Chemical, St. Louis, MO, USA) and 0.1% SDS for 60 min at 65 °C. After proteinase K treatment, membranes were washed with 1% SDS, 5x standard saline citrate (SSC) and then incubated in 50% formamide (Sigma Chemical), 10 mM sodium phosphate, pH 6.5, at 65 °C for 60 minutes.

Stripping efficiency was evaluated by rinsing the membranes, adding fresh conjugate and developing with LumiPhos 530 substrate. Membranes were then rinsed and re-probed with a single probe concentration (8 ng/mL).

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Results

The initial probing shows reduced sensitivity as probe concentration is reduced. After stripping with proteinase K and formamide, most of the signal was regenerated without adding new probe. After alkaline stripping, no signal was observed. The alkaline-stripped membranes were rinsed and re-probed with a single probe concentration. After incubation with conjugate and substrate, signal was regenerated with no loss of sensitivity. This process was repeated for a total of five times. (For complete data, reference Dubitsky, A, and DeFiglia, JA. Stripping of Digoxigenin-labeled Probes from Nylon Membranes. BioTechniques, 1995: 19: 210-212.)

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Discussion

Incubation with fresh conjugate and substrate can be used to establish complete removal of probe DNA from target DNA immobilized on a membrane. New results with proteinase/formamide-stripped membranes show this clearly; after proteinase and formamide treatment, signal was virtually as strong as before stripping. This is in contrast to the NaOH treatment, which resulted in no signal when conjugate and substrate were reapplied.

Experiments in our laboratory in 1988 indicated that NaOH stripping methods sometimes resulted in a loss of target DNA after repetitive stripping events. Blots used in the experiments described here showed little or no loss of sensitivity after five probings.

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Conclusions

Some researchers have reported difficulties in stripping DIG-labeled probe after hybridization. We have confirmed that stripping with formamide, with or without proteinase K pretreatment, is ineffective for removing DIG-labeled probe. Reducing the probe concentration (and resulting signal level) did not increase stripping effectiveness. The proteinase K treatment digested proteins on the membrane, including blocking agents and the alkaline phosphatase conjugate, while leaving the hybridized probe in place.

Stripping these membranes with 0.2 M NaOH, 0.1% SDS was effective in removing alkali stable DIG-labeled probe. This was demonstrated by the addition of fresh conjugate and substrate. There was no detectable signal after any of our NaOH stripping events.

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References

Boehringer Mannheim Biochemicals. 1992. The Genius System User's Guide for Filter Hybridization Version 2.0. Indianapolis, IN. p.66.
Dubitsky, A., J. Brown and H. Brandwein. 1992. Chemiluminescent detection of DNA on nylon membranes. BioTechniques 13:392-400.
Moller, E., A. Schilling and P. Karlovsky. 1994. Stripping of hybridization membranes. BioTechniques 17:682-684.

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