Chemical Biology: Bioconjugation and Reporting on a Molecule in a Cell · seed 1 · A4, ink-friendly. The answer key prints on its own page for grown-ups.

Tagging one protein inside a living cell

Science · Chemistry · ages 23-24
Name ______________________   Date ____________

  1. A reagent reacts with accessible native thiols and has no other targeting mechanism. What limits its use for labelling one protein?

    • It must recognise only the intended protein even when others expose thiols
    • It cannot react with any accessible thiol in an aqueous environment
    • It can label other proteins with accessible thiols

  2. You plan to label a protein in a living cell's near-neutral cytosol. Why choose chemistry that works under those conditions?

    • To keep the assay compatible with living cells
    • To make every fluorescent reporter emit the same colour
    • To stop all native amines and thiols from reacting

  3. A position-only fluorescent tag can show where labelled protein is detected, but its brightness alone does not establish catalytic activity.

    Circle one:   True   False

  4. Which comparison best tests whether fluorescence depends on an installed handle?

    • Handle-bearing cells given extra dye, compared with cells imaged using a dimmer lamp
    • Handle-bearing cells without washing, compared with cells given a different reporter
    • Matched cells with and without the handle, labelled and imaged under the same conditions

  5. A live-cell assay uses a low reagent concentration and a short labelling period. Which reaction property must you check?

    • Slow reaction throughout the available labelling period
    • Adequate rate at the chosen concentration and conditions
    • High rate measured only in hot, strongly acidic solvent

  6. Many proteins expose native thiols. You can install a unique handle on the target. Which plan offers a more selective route, subject to experimental checks?

    • Increase the dose of a reagent that reacts with accessible thiols on many proteins
    • Pair the target handle with a reagent that avoids native groups
    • Use a brighter general thiol label without changing how it recognises proteins

  7. In a hypothetical matched assay, handle-bearing cells give 8 fluorescence units and no-handle cells give 2. Dose, washing and imaging settings are the same. Which conclusion is supported?

    • Background is present; the higher signal supports handle dependence
    • Every fluorescent label is on the target because the no-handle control is dimmer
    • The no-handle signal proves that neither sample contains any labelled target protein

  8. You want to measure changes in a protein's catalytic activity over time. Which reporter directly addresses that question?

    • An activity-sensitive reporter calibrated against that activity
    • A position-only tag observed without any link between its signal and catalytic activity
    • A brighter position-only tag assumed to measure catalysis from brightness alone
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Answer key

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

Tagging one protein inside a living cell W1-mt_nsNwVhPRFN-s1

  1. It can label other proteins with accessible thiols · Thiol reactivity recognises a chemical group, not a protein's identity. Other proteins with accessible thiols can receive labels too.
  2. To keep the assay compatible with living cells · The reaction should work in the target compartment without imposing incompatible conditions. Near-neutral pH does not set every dye's colour or prevent every native-group reaction.
  3. True · A position-only tag gives a location readout. Catalytic activity requires a readout linked to that activity, rather than brightness alone.
  4. Matched cells with and without the handle, labelled and imaged under the same conditions · The matched comparison changes handle presence while keeping labelling and measurement conditions comparable. It tests handle dependence, not complete target specificity.
  5. Adequate rate at the chosen concentration and conditions · The rate must give useful labelling within the actual assay time. A rate measured only under incompatible conditions does not establish suitability for this assay.
  6. Pair the target handle with a reagent that avoids native groups · A matched handle-reagent pair adds a source of selectivity. Handle placement, reagent access, off-target labelling and target function still need checking.
  7. Background is present; the higher signal supports handle dependence · The 2-unit no-handle signal reveals handle-independent background. The higher signal with the handle supports a handle-dependent contribution, but does not identify every labelled molecule.
  8. An activity-sensitive reporter calibrated against that activity · The readout must respond to the defined activity and be calibrated against it. Location or increased brightness alone does not supply that relationship.
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