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2 Jun 2019

Explain how protein purification occurs vis differential and rate zonal centrifugation.

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3 Jun 2019

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Differential centrifugation separates cell components according to

	Mass
	Sedimentation rate

	Density
	Charge

Density gradient centrifugation separates cell components according to

	Density
	Sedimentation rate

	Mass
	Charge

If you are presented with a graph with many fractions on the x-axis, or a western blot where the lanes were from many fractions, what method was used?

	Affinity purification
	Western blot

	Density gradient centrifugation
	Differential centrifugation

What is the principle behind denaturing protein gel electrophoresis?

	Proteins are separated according to type of amino acid R groups
	Proteins are separated according to charge

Proteins are separated according to size

In denaturing gel electrophoresis, proteins that run farther down the gel are:

	Smaller than those at the top of the gel
	Larger than those at the top of the gel

What is a western/immunoblot?

	All proteins are stained with a general protein stain
	Specific proteins are identified by antibodies

Specific proteins are purified and detected by a general protein stain

What is the principle behind affinity purification?

	Specific molecules are removed from solution by a high affinity interaction on a solid support
	Specific molecules have an exclusive sedimentation rate that be exploited with differential centrifugation

Specific molecules have an exclusive size that can be exploited with SDS-PAGE

Which of the following is quantification of variance in an experiment?

	Median
	Error bars

Mean

In lab, our objective was to obtain a mitochondrial enriched extract from cauliflower, use the mitochondrial enzyme succinate dehydrogenase as a marker for mitochondrial enrichment, and provide a better understanding of the activity of enzymes using succinate dehydrogenase as an example.

Part 1 was: Enrichment of mitochondria from cauliflower extract by differential sedimentation

Part 2 was: Monitoring of fractions for specific activity of the mitochondrial enzyme succinate dehydrogenase.

What is the general differential centrifugation scheme you perform in the differential centrifugation and SDH Activity lab?

A scientist recently identified a nuclear protein involved in nuclear lamina assembly. Which fraction of your differential centrifugation scheme would be the most enriched fraction for your protein of interest? Why?

How did you detect the enrichment of mitochondria in the isolated fractions?

Which of the fractions you collected should contain the highest enrichment of mitochondria? Does your specific activity of your fractions support this?

In order to calculate the specific activity of the isolated fractions, the protein concentration has to be determined. Why is the protein concentration necessary for accurate comparison of the enzyme activity of the fractions?

Protein Purification: You have a tube containing trypsinogen, chymotrypsinogen and chymotrypsin in an aqueous buffer. Using atleast two methods of protein purification , propose a scheme for seperating the three proteins. Explain how each method works. physically or chemically. If you choose affinity chromatography, you must clearly explain your choice of affinity substrate.

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Explain how protein purification occurs vis differential and rate zonal centrifugation.

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