Fractionation, Blotting and Pulling Down a Binding Partner · seed 1 · A4, ink-friendly. The answer key prints on its own page for grown-ups.
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Spin it, blot it, pull it down

Science · Biochemistry & Molecular Biology · ages 21-22
Name ______________________   Date ____________

  1. A blot band is dark. What does that mean after checking the loading control?

    • The protein weighs exactly that many grams
    • The protein is present in a rough amount
    • The antibody has failed completely

  2. A gentle first spin drops one organelle. Which one?

    • Nuclei
    • Mitochondria
    • Ribosomes

  3. A pull-down hit proves the two proteins touch each other directly.

    Circle one:   True   False

  4. What are the three stages of a Western blot?

    • Fix cells, add GFP tag, film over time
    • Spin nuclei, spin mitochondria, spin ribosomes
    • Separate on gel, transfer to membrane, probe with antibody

  5. Why does SDS let a gel sort by size alone?

    • SDS dyes every protein a different colour
    • SDS cuts all proteins to equal length
    • Detergent equalizes charge so only size decides speed

  6. What does the loading control correct for?

    • Wrong antibody colour
    • Uneven filling of the lanes
    • Too much microscope zoom

  7. A brief binding partner never shows in a pull-down. Why?

    • Beads only grab DNA
    • Weak partners wash away before reading
    • Gels cannot show small proteins

  8. One lane is empty but its loading control band is healthy. What do you conclude?

    • The gel ran backwards
    • The control proves the protein is there
    • The protein is absent or vanishingly low
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Answer key

For grown-ups. Fold this page away before handing over the rest.

Spin it, blot it, pull it down W1-mt_MRfFQlBIPy-s1

  1. The protein is present in a rough amount · Darkness reports rough amount once uneven filling is corrected.
  2. Nuclei · Biggest and densest sink at the lowest speed.
  3. False · They may share a third partner in one complex instead.
  4. Separate on gel, transfer to membrane, probe with antibody · Size sorting plus antibody identity gives high confidence.
  5. Detergent equalizes charge so only size decides speed · With charge uniform, the mesh holds back only big chains.
  6. Uneven filling of the lanes · Divide each band by its control before comparing amounts.
  7. Weak partners wash away before reading · Washing keeps strong binders and loses fleeting ones.
  8. The protein is absent or vanishingly low · A working control with no target band means nothing to find.
Worksheet · LightMySky