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Optimization of blocking conditions for fluorescent Western blot
1Leibniz Institute on Aging, Fritz Lipmann Institute, 07745, Jena, Germany.
Analytical Biochemistry
|February 5, 2020
Summary
Optimizing blocking conditions is key for fluorescent Western blots (WB). This study clarifies blocker-induced autofluorescence and provides guidance for effective, cost-efficient blocking protocols to improve signal detection.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunodetection Techniques
Background:
- Blocking is crucial for reducing background in Western blots (WB).
- Optimal blocking conditions for fluorescent WB are less established, with blockers potentially causing autofluorescence.
- Autofluorescence from Tween 20 is negligible when wet but significant when dry.
Purpose of the Study:
- To systematically investigate and clarify blocking conditions for fluorescent Western blots.
- To evaluate the impact of various blockers and conditions on autofluorescence and signal detection.
- To provide a reference for cost-effective blocking strategies in fluorescent WB.
Main Methods:
- Systematic investigation of blocking conditions, including blockers, imaging configurations, membranes, and hydration (wet/dry).
- Evaluation of autofluorescence from Tween 20 under different conditions.
- Assessment of common blockers (milk, BSA, fish gelatin, PVP) for autofluorescence, blocking efficiency, and cost.
- Testing the effect of sodium dodecyl sulfate (SDS) on non-specific bands and background.
Main Results:
- Autofluorescence is influenced by a combination of factors: blocker, excitation/emission settings, membrane, and hydration state.
- Tween 20 autofluorescence is minimal when wet but can be significant when dried.
- Different blockers (milk, BSA, fish gelatin, PVP) present unique trade-offs regarding autofluorescence, efficiency, and cost.
- Inclusion of low concentrations of SDS (0.005%-0.02%) can reduce non-specific binding but may also decrease specific signals.
- Pre-testing antibodies may be necessary to select the most suitable blocker for optimal performance.
Conclusions:
- Autofluorescence in fluorescent WB is multifactorial and highly dependent on specific experimental conditions.
- Careful selection of blocking agents and conditions is essential to minimize background and maximize specific signal detection.
- This study offers valuable insights for establishing cost-effective and efficient blocking protocols for fluorescent Western blotting.
