Fractionation, Blotting and Pulling Down a Binding Partner
Breaking cells open and spinning them at increasing speeds separates organelles by size and density, and a blot then asks whether a named protein is present in a fraction. Precipitating one protein with an antibody brings its partners with it, which is how binding is tested.
What a learner can do afterwards
- Predicts which fraction an organelle appears in from a centrifugation scheme.
- Reads a blot as presence, absence and rough amount, with a loading control taken into account.
- Explains why a pull-down result can be a real partner or a shared third partner.
1 · Read
Cells are taken apart gently so each organelle can be studied alone. Homogenising cracks the membrane while leaving organelles whole. Spins of rising speed then drop nuclei first, mitochondria next, and ribosomes last. The scheme works because bigger and denser parts sink at lower speeds. Each pellet is a rough cut that later steps purify further.
A Western blot asks whether one specific protein sits in a messy mixture. Gels first sort proteins by size: SDS coats them evenly in charge, so small chains race far while big ones crawl, read against a known ladder. The blot transfers them to a membrane, and an antibody finds only the target. Band position gives size and darkness gives rough amount.
A pull-down tests binding with a bait. Beads hold one protein and keep whatever grips it through the washes, and the bound set is read on a gel. A hit proves shared company, not direct contact: the partner may bind the bait directly, or both may cling to one shared third protein in the same complex. Weak or brief partners wash away and get missed.
Always divide by the loading control before judging amount, since it corrects for uneven filling. An empty control lane voids the whole blot. No band beside a healthy control lane means absent or vanishingly low. Predict each pellet from the spin speed before you open the tube.
Gentle spins sort organelles by size, blots ask if one protein is there and how much, and pull-downs catch binders direct or indirect.
2 · Watch
Take it off screen
Where it sits
8 questions wait behind this lesson, each with its answer explained. Every answer feeds the sky: stars light as they are learned, and dim when it is time to come back.