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For 6 Blots. Time Required: Electrophoresis & Blot Preparation: 60 - 90 min. Blot: overnight. Detection: 45 min.
The objective of this experiment is to understand the molecular biology and pathogenesis of AIDS. Students use the Western Blot method to test for the presence of simulated viral proteins in cell culture extracts from hypothetical patients who have "tested positive" for HIV-1 by ELISA. Cell proteins are separated by horizontal agarose gel electrophoresis, transferred to a membrane, and detected by staining. This experiment does not require an electrotransfer apparatus.
REQUIREMENTS: Horizontal gel electrophoresis apparatus; D.C. power supply; microwave oven, hot plate, or Bunsen burner; automatic micropipets (0-50 µl) and tips (optional); beakers, flasks, and graduated cylinder; paper towels; methanol (95-100%); glacial acetic acid. Some components require refrigerator storage.
CONTENTS: Positive & Negative Control Samples; Patient Samples; Protein Agarose; Tris-Glycine-SDS Electrophoresis Buffer; Tris-Glycine Buffer for Gel Preparation; PVDF Membrane, Filter Paper; Protein Visualization Stain; Filter Paper; Complete instructions; background information; and study questions.