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#protease digestion

2 public questions tagged with this topic.

Blotting Techniques

To detect expression of a protein in mitochondria after protease digestion, you use:

Proving internal mitochondrial localization requires protease protection assay combined with protein detection that provides molecular weight information. Isolated mitochondria are treated with proteinase K; outer membrane proteins degrade while matrix and inner membrane proteins protected by intact membranes remain. After lysis, samples resolved by SDS-PAGE and probed immunologically confirm persistence of target. Reverse transcription PCR measures RNA absent after protease treatment assessment, Southern detects DNA, and ELISA quantifies without size verification, unable to distinguish trunca

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Fluorescence and CD Spectroscopy

CD change upon protease digestion shows:

Protease hydrolysis cleaves peptide bonds, fragmenting chain and disrupting hydrogen bonded networks that stabilize helices and sheets. Far-ultraviolet circular dichroism monitors these networks via amide n to pi-star and pi to pi-star bands; loss of distinct minima at 208, 222 or 218 nanometers and appearance of random coil signature near 195 nanometers reflects conformational alteration and unfolding of remaining fragments. Primary sequence composition change alone without structural rearrangement would not alter CD significantly; fluorescence quenching reports side chain environment, disulf

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details