week 6 EXPLORING PROTEINS purification and locolisation

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12 Terms

1
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Why is protein purification important?

Essential for biopharmaceutical production, research, and clinical studies; reduces reliance on animal sources.

2
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What is the capture phase in protein purification?

Cell/tissue lysis and isolation of the protein from crude extract using mechanical, enzymatic, or detergent methods.

3
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What happens during the intermediate purification phase?

Removal of impurities by centrifugation and precipitation; characterization of protein properties.

4
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What is salting out in protein purification?

Precipitation of proteins by ammonium sulfate based on solubility differences.

5
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Describe the polishing phase.

High purity achieved through chromatography (size-exclusion, ion-exchange, affinity); use of protein tags like His-tags.

6
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Name two methods to test purified protein functionality.

ELISA for quantification/identity and Western Blotting for protein presence and integrity.

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Why is protein localisation important?

Crucial for proper cellular function; mislocalisation can cause diseases like Parkinson’s and Alzheimer’s.

8
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What are signal sequences?

Short amino acid motifs directing proteins to specific compartments (e.g., nucleus, mitochondria, ER).

9
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How does the endoplasmic reticulum (ER) assist protein targeting?

ER signal sequences are recognized by SRP, directing proteins to the ER for processing.

10
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What is immunofluorescence?

A method using antibodies to stain fixed cells and visualize protein location with high specificity.

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How does GFP tagging help in protein localisation?

Fusion of GFP allows live cell imaging of protein localisation and dynamics in real time. and no need for external dyes or substrates, GFP fluoresces naturally when expressed, simplifying experiments.

Versatile and genetically encoded: Can be fused to almost any protein, allowing broad application across different cell types and organisms.

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What are FRAP and FRET used to study?

FRAP studies protein mobility by photobleaching fluorescence; FRET detects protein-protein interactions by energy transfer.