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V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting
Published on: December 30, 2013
Direct Blue 71 staining as a destaining-free alternative loading control method for Western blotting
Li Zeng1, Jing Guo, Hong-Bo Xu
1Department of Neurology, First Affiliated Hospital, Chongqing Medical University, Chongqing, PR China.
Electrophoresis
|May 29, 2013
Summary
Direct Blue 71 (DB71) staining offers a reliable, destaining-free loading control for Western blotting. This novel method is equivalent or superior to traditional techniques for neuroscientific samples.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Western blotting requires loading controls to normalize protein levels.
- Traditional methods include housekeeping proteins and total protein staining.
- Direct Blue 71 (DB71) is a novel dye-binding stain compatible with immunodetection.
Purpose of the Study:
- To evaluate DB71 staining as a loading control for Western blotting.
- To compare DB71 with traditional methods using neuroscientific samples.
- To assess DB71's impact on assay reliability and dynamic range.
Main Methods:
- DB71 and Coomassie Brilliant Blue (CBB) staining were used.
- Housekeeping proteins and proteins of interest were analyzed.
- Reliability, repeatability, and linear dynamic range were assessed across protein loads (2.5-40 μg).
- Neuroscientific samples included human plasma, human oligodendrocytes, and rat brain.
Main Results:
- DB71 demonstrated equivalent or superior reliability, repeatability, and linear dynamic range compared to CBB and housekeeping proteins across all sample types.
- DB71 staining did not negatively affect the reliability, repeatability, or linear dynamic range of subsequent immunoreactions.
- DB71 proved effective in human plasma, human oligodendrocytes, and rat brain samples.
Conclusions:
- DB71 staining is a viable and effective loading control for Western blotting.
- DB71 offers a destaining-free alternative to traditional loading control methods.
- This method is suitable for various neuroscientific sample types.
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01:15Western Blotting
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The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
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Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...