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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?

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Keith Leannon
Keith LeannonLv2
31 Jul 2019

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Related questions

Biochemistry Protocols: Your First Protein Purification

As the newest student in a biochemistry research lab, you are given responsibility for purifying a mitochondria protein. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answers.

(a) You pick up 20 kg of beef hearts from a nearby slaughterhouse. You transport the hearts on ice, and perform each step of the purification on ice or in a walk-in cold room. You homogenize the beef heart tissue in a high-speed blender in a medium containing 0.2 M sucrose, buffered to a pH of 7.2.

Why do you use beef heart tissue, and in such large quantity?

What is the purpose of keeping the tissue cold and suspending it in 0.2 M sucrose, at pH 7.2? What happens to the tissue when it is homogenized?

(b) You subject the resulting heart homogenate, which is dense and opaque, to a series of differential centrifugation steps. What does this accomplish?

(c) You proceed with the purification using the supernatant fraction that contains mostly intact mitochondria. Next you osmotically lyse the mitochondria. The lysate, which is less dense than the homogenate, but still opaque, consists primarily of mitochondrial membranes and internal mitochondrial contents. To this lysate you add ammonium sulfate, You centrifuge the solution, decant the supernatant, and discard the

pellet. To the supernatant, which is clearer than the lysate, you add more ammonium sulfate. Once again, you centrifuge the sample, but this time you save the pellet because it contains

the protein of interest. What is the rationale for the two-step addition of the salt?

(d) You solubilize the ammonium sulfate pellet containing the mitochondrial proteins and dialyze it overnight against large volumes of buffered (pH 7.2) solution. Why isn’t ammonium sulfate included in the dialysis buffer? Why do

you use the buffer solution instead of water?

(e) You run the dialyzed solution over a size-exclusion chromatographic column. Following the protocol, you collect the first protein fraction that exits the column, and discard the rest of the fractions that elute from the column later. You detect the protein by measuring UV absorbance (at 280 nm) in the fractions. What does the instruction to collect the first fraction tell you about the protein? Why is UV absorbance at 280 nm a good way to monitor for the presence

of protein in the eluted fractions?

(f) You place the fraction collected in (e) on a cation exchange chromatographic column. After discarding the initial solution that exits the column, you add a washing solution of higher pH to the column and collect the protein fraction that immediately elutes. Explain what you are doing.

(g) You run a small sample of your fraction, now very reduced in volume and quite clear (though tinged pink), on an isoelectric focusing gel. When stained, the gel shows three sharp bands. According to the protocol, the protein of interest is the one with the pI of 5.6, but you decide to do one more assay of the protein’s purity. You cut out the pI 5.6 band

and subject it to SDS polyacrylamide gel electrophoresis. The protein resolves as a single band. Why were you unconvinced of the purity of the “single” protein band on your

isoelectric focusing gel? What did the results of the SDS gel tell you? Why is it important to do the SDS gel electrophoresis after the isoelectric focusing.

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