= Protease protection assay
A <protease protection assay> asks whether a barrier prevents an added <protease> from reaching a <protein>. A membrane-disruption comparison tests whether protection depends on that barrier.
\Image[/protein-targeting-protease-protection-controls.png]
{title=<Protease protection assay>: accessibility and control logic}
{description=Original qualitative decision diagram for a completed soluble cargo. Icons and words describe expected outcomes under stated assumptions; they are not an <autoradiography> readout, copied historical lanes, measured survival fractions or simulated experimental intensities. <Detergent> is added after the import reaction. The historical <nascent chain> experiment also retained small <ribosome>-protected fragments, which this completed-cargo model deliberately distinguishes.}
The membrane-disruption test was performed by <David D. Sabatini> and <Günter Blobel> in 1970. Rat-liver <microsomes> purified by <sucrose> <centrifugation> incorporated radioactive <leucine> during <cell-free translation>. Samples received cold <trypsin> and <chymotrypsin>, with or without membrane-solubilizing <detergents>. <Electron microscopy> checked vesicle preservation; <size-exclusion chromatography> separated protected fragment sizes. Large membrane-associated fragments lost protection after disruption, whereas small <ribosome>-protected fragments persisted. An exposed <ribosome> preparation remained digestible when mixed with <microsomes>, checking that membranes did not simply inhibit the <proteases>.<Controlled Proteolysis of Nascent Polypeptides in Rat Liver Cell Fractions II by David D. Sabatini and Günter Blobel>[Controlled proteolysis of nascent polypeptides in rat liver cell fractions. II, Methods p. 147; activity control p. 148; Figure 5C–D and text p. 152; Discussion pp. 155–156]{ref}
To read the qualitative diagram, first compare untreated aliquots: they establish that detectable cargo exists before digestion. Next compare external <protease> with intact versus disrupted membranes. Persistence only while the barrier remains intact supports enclosure, provided the exposed-cargo control confirms active <protease> in both chemical conditions. The <detergent>-only aliquot checks that disruption itself does not destroy recovery or detection. Adding membranes after synthesis tests a different alternative: capture of a completed product rather than entry during <biological translation>.
This is an argument from controlled accessibility. Binding predicts membrane association but generally leaves an exposed cargo digestible. Enclosure predicts protection that depends on membrane integrity. However, <protein aggregation>, intrinsic resistance to <proteolysis>, inactive <protease> or undetected small fragments can obscure this distinction. For a <transmembrane protein>, expect protection of the enclosed portion rather than necessarily the full chain. Combine fragment size, compartment controls and <membrane topology>; a protected signal alone does not uniquely specify the whole <protein>'s location.
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