SOP Generator

Document ID: LAB-PRT-004

Step Step Name Task Description Caveats Visual Aid
SECTION 1: Exosome Lysis
1 Resuspend Pellet Resuspend exosome pellet in 100 µL of cold RIPA buffer. This buffer's detergents break open the exosome membrane [3], releasing proteins.
  • Keep on ice to prevent degradation.
Scientist working with samples
2 Incubate & Lyse Incubate on ice for 20 minutes, vortexing briefly every 5 minutes.
  • Gentle vortexing is sufficient.
  • Wear appropriate PPE [2].
3 Pellet Debris Centrifuge at 14,000 x g for 10 mins at 4°C. This pellets dense, insoluble components [8], purifying the lysate in the supernatant.
  • Balance centrifuge.
  • Carefully aspirate supernatant.
SECTION 2: DNA Removal
4 Add DNase Transfer supernatant to a new tube and add 1 µL of DNase I, an enzyme that degrades DNA [4].
  • Use RNase-free DNase I.
  • Mix by gentle flicking.
  • Inactivate DNase later with Proteinase K [5].
Petri dishes with cultures
5 Incubate Incubate at 37°C for 15 minutes to ensure optimal enzymatic activity [4].
  • Do not exceed incubation time.
SECTION 3: Sample Preparation
6 Add Sample Buffer Add Laemmli buffer to 15 µg of protein. The buffer's SDS coats proteins with a negative charge for separation [7].
  • Accurate quantification is critical [1].
Test tubes in a rack
7 Denature Heat sample at 95°C for 5 minutes to linearize proteins for accurate SDS-PAGE separation [6].
  • Use a heated lid to prevent evaporation.
  • Briefly spin down after heating.
8 Load Gel Load the entire sample into a well of a 4-20% Tris-Glycine gel.
  • Load slowly for a sharp band.
SECTION 4: Analysis
9 Analyze Gel Analyze the finished gel on the Gel Electrophoresis Analyzer to determine protein concentrations [9].
  • You will need to upload a photo of the SDS-Page Gel.