✓Prepare sucrose density gradients for ribosome separation
✓Extract translating ribosomes from yeast cells
✓Centrifuge gradients and collect ribosome fractions
✓Interpret polyribosome profiles to assess translation regulation
Protocol
Biopharma Insights This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.
Difficulty
advanced
Total time
~6-8 hours per sample
Model organism
Saccharomyces cerevisiae
Biosafety
BSL-1
Steps
1
Prepare sucrose density gradients
Create sucrose gradients of increasing concentration in centrifuge tubes to enable separation of ribosomal populations during centrifugation.
▶ 01:15
2
Prepare extracts from yeast cells
Lyse yeast cells and isolate the cytoplasmic extract containing translating ribosomes, ensuring conditions preserve the polyribosome integrity.
▶ 02:53
3
Centrifuge gradients and collect fractions
Apply the extract onto the sucrose gradient, centrifuge to separate monosomes and polyribosomes, and then fractionate while monitoring absorbance.
▶ 05:06
4
Representative trace of ribosome extract
Display and interpret a typical absorbance profile showing peaks corresponding to monosomes and polyribosomes.
▶ 08:35
💬 Comments coming soon
New protocols and pitfalls, in your inbox
A short email when we add notable lab videos and failure cases. No spam, unsubscribe anytime.