Home › Microbiology › Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis
Microbiology JoVE (Open Access) Citable · DOI

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

DOI: 10.3791/838-v
What you'll learn
  • ✓ Prepare semi-denaturing detergent-agarose gels for amyloid polymer detection
  • ✓ Execute sample preparation and loading for SDD-AGE analysis
  • ✓ Perform capillary transfer and interpret amyloid aggregation patterns
  • ✓ Scale SDD-AGE methodology for high-throughput screening applications
Protocol

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Difficulty
intermediate
Total time
~4-6 hours per batch (gel preparation through transfer)

Steps

1
Understand SDD-AGE principle and applications

Review the fundamentals of semi-denaturing detergent-agarose gel electrophoresis for detecting and characterizing amyloid-like polymers in cellular systems. Understand how this technique enables large-scale screening applications.

▶ 00:48
2
Prepare semi-denaturing detergent-agarose gel

Assemble and cast the agarose gel with appropriate buffer and detergent composition according to protocol specifications. Allow gel to polymerize completely before sample loading.

▶ 01:41
3
Prepare and load cell samples

Lyse cells and prepare samples under semi-denaturing conditions, then load onto the prepared gel ensuring proper sample distribution across lanes.

▶ 03:10
4
Execute capillary gel transfer

Transfer separated polymers from gel to membrane using capillary action-based methodology to preserve amyloid aggregate integrity and resolution.

▶ 06:23
5
Analyze and interpret blotting results

Visualize transferred amyloid-like polymers and characterize banding patterns to assess aggregation status and polymer size distribution in samples.

▶ 09:15
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