---
title: "Fractionation, Blotting and Pulling Down a Binding Partner"
description: "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 wi"
canonical: https://lightmysky.com/learn/science/fractionation-blotting-and-pulling-down-a-binding-partner-mt_MRfFQlBIPy
source: https://lightmysky.com/learn/science/fractionation-blotting-and-pulling-down-a-binding-partner-mt_MRfFQlBIPy.md
retrieved: 2026-09-12
---

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# 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.

Subject: Science · Area: Biochemistry & Molecular Biology · Ages 21 to 22
Page: https://lightmysky.com/learn/science/fractionation-blotting-and-pulling-down-a-binding-partner-mt_MRfFQlBIPy

## Ready when they can

- 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.

## Lesson: Spin it, blot it, pull it down

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.

**Tip.** 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.

**Recap.** Gentle spins sort organelles by size, blots ask if one protein is there and how much, and pull-downs catch binders direct or indirect.

## Practice

8 questions on this page, each with its working shown.

## Needs first

- [Fluorescent Tagging and Reading Cell Images](https://lightmysky.com/learn/science/fluorescent-tagging-and-reading-cell-images-mt_J0toch-ZZ3)
- [PCR, Electrophoresis and DNA Profiling](https://lightmysky.com/learn/science/pcr-electrophoresis-and-dna-profiling-mt_Sv1jV0XnsX)

## Opens up

- [Proteomics: Mass Spectra, Quantification and Interaction Maps](https://lightmysky.com/learn/science/proteomics-mass-spectra-quantification-and-interaction-maps-mt_a-8UHB2SFR)
- [Designing a Biological Experiment: Units, Controls and Randomisation](https://lightmysky.com/learn/science/designing-a-biological-experiment-units-controls-and-randomisation-mt_Hc6BAdlkLf)