lec14-15 Flashcards
What does centrifugation separate macromolecules based on?
Centrifugation separates macromolecules based on their density, centrifugal force (multiples of g), and centrifugation time (T).
What are the key steps in differential centrifugation for protein purification?
Step 1: Centrifuge at 1,000 x g for 5 minutes to isolate the nuclear fraction. and then resuspend the pellet in a seprate test tube (most dense)
Step 2: Centrifuge at 10,000 x g for 10 minutes to isolate the mitochondrial fraction.
Step 3: Centrifuge at 15,000 x g for 30 minutes to isolate the membrane fraction.
Final Step: The remaining supernatant contains the cytosol fraction.
What is “salting out” used for in protein purification, and how does it work?
“Salting out” is a technique used to selectively precipitate proteins from a solution by adding high concentrations of salt (like ammonium sulfate). The salt competes with proteins for water molecules, decreasing the proteins’ solubility, leading to aggregation and precipitation. It’s often employed as an initial step to concentrate or purify a target protein. diff proteins have diff solubility eg. fibrinogen presips at 0.8M ammonium sulfate but serum albumin at 2.4M
✨ Affinity Chromatography ✨
What it does: separates proteins based on specific binding to a ligand
How it works: the target protein sticks to a ligand on the resin, while others wash away
How to get the protein back: change pH, add a competing molecule, or use salt to break the bond
Example: using an antibody to catch a specific protein in a mixture
it’s like a VIP pass—only the right protein gets in, and everything else is left out!
✨ Gel Chromatography
What it does: separates proteins based on size
How it works:
Big proteins can’t enter gel bead pores → move fast → come out first
Small proteins enter the pores → take longer → come out last
Example: sorting balls through holes—big ones roll straight through, small ones get stuck and take longer!
✨ Ion-Exchange Chromatography ✨
What it does: separates proteins based on charge
How it works:
Proteins with opposite charge stick to the resin
Other proteins flow through
How to release bound proteins:
Add salt → ions compete & knock proteins off
Change pH → alters protein charge so it lets go
column chromatography (3)
🔹 Affinity Chromatography – Specific binding
Target protein binds to a ligand on the resin
Release: change pH, add competitor, or use salt
🏆 VIP pass – only one protein gets in!
🔹 Gel Filtration (Size Exclusion) – Size-based separation
Big proteins elute first, small ones get trapped in beads
🏀 Big balls roll through fast, small ones get stuck!
🔹 Ion-Exchange Chromatography – Charge-based separation
Opposite charges attract, others wash out
Release: add salt or change pH
🧲 Like magnets – the right charge sticks!
what is SDS-PAGE and what property of proteins does it separate them by?
SDS-PAGE (PolyAcrylamide Gel Electrophoresis) is a technique used to separate proteins based on their mass. Proteins are given a uniform negative charge by association with SDS (sodium dodecyl sulfate), and an electric field is applied to move the proteins through a gel matrix, with smaller proteins migrating faster than larger ones.
Proteomics
Definition: The large-scale study of proteins, focusing on their structure, function, and interactions.
