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Flashcards in Quiz 2 Deck (21)
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1

- Cell fractionation is...
- by either...

- ...a general procedure that consists of disrupting the cell membrane (and cell wall of plants) to release the cell contents and then separate the organelles based upon differences in size or density
- ...differential centrifugation or density gradient centrifugation.

2

differential centrifugation = the homogenate is...

= the homogenate is centrifuged repeatedly at successively higher speeds, sedimenting progressively smaller particles.

3

density gradient centrifugation is used for

...the best resolution of cell fractions and the least contamination by extraneous organelles

4

density gradient centrifugation = the homogenate is...
- As the tube spins...
- The topmost layer contains...
- Afterwards...

= the homogenate is added to the top of a centrifuge tube that contains a sucrose gradient in which the most concentrated (most dense) sucrose solution is at the bottom of the tube and the least concentrated (least dense) sucrose solution is at the top.
- ...the organelles distribute themselves in the sucrose gradient by size, the largest organelles sedimenting most rapidly
- ...the cytosol
- ...the cell fractions are removed by puncturing the bottom of the tube and allowing the contents in each layer to drip into a separate test tube

5

The sedimentation coefficient =

= a physical constant frequently encountered in biological literature

6

A particle’s sedimentation coefficient(s) is determined from...

...its rate of sedimentation under conditions of unit acceleration.

7

The factors that determine how rapidly a particle sediments in a centrifugal field are: (2)

1.) the particle’s radius
2.) its effective density (the difference between its density and the density of the liquid through which it is moving)

8

In general, the _____ the particle, the ______ its sedimentation coefficient will be

larger
greater

9

The force generated by the centrifuge is expressed as...
With units...

...a relative centrifugal force (RCF)
...“g” being the force of the earth’s gravity exerted upon a mass

10

RCF =

= a function of the speed of centrifugation in revolutions per minute (rpm) and the distance of the particle from the axis of rotation.

11

the purpose of an cold isotonic homogenization buffer in the fractionation of chloroplasts and mitochondria = (3)

1.) cold to stop enzyme reactions/maintain the integrity of the organelles and enzymes during the fractionation procedure
2.) isotonic to stop osmosis
3.) buffer to stop pH changes

12

isoelectric point (pI) =

the pH at which a particular molecule carries no net electrical charge or is electrically neutral

13

At a pH below their pI, proteins carry...
At a pH above their pI, proteins carry...

...a net positive charge
...a net negative charge.

14

- @ its pI, proteins are at their...
- Therefore, by changing the pH of the solution to the pI, the...

...lowest solubility (due to net 0 charge)
...solubility of the protein will be lowered

15

- Ammonium sulfate precipitation allows for...
- The solubility of a protein is dependent on...

...the isolation of proteins by altering their solubility
...the salt concentration

16

- At low salt concentrations...
- This is commonly known as...

...the presence of salt stabilizes the various charged groups on a protein molecule, thus attracting the protein into the solution and enhancing the solubility of protein.
...salting-in.

17

- As the salt concentration is increased...
- Further increases in the salt concentration result in...
- Finally, the protein starts to...
- This phenomenon of protein precipitation in the presence of excess salt is known as...

... a point of maximum protein solubility is usually reached
...fewer water molecules available to solubilize protein (since the water molecules are being used to solubilize the salts)
...precipitate when there is not a sufficient number of water molecules to interact with (solubilize) the protein molecules
...salting-out.

18

the role of temperature in fractionation of cellular components such as chloroplasts, mitochondria and proteins =

cold to stop enzyme reactions/maintain the integrity of the organelles and enzymes during the fractionation procedure

19

why protein quantification is assessed =

= in order to normalize (“make equal”) the amount of starting material for subsequent assays
& allows us to know the protein concentrations of our samples, so that we can adjust the volumes for subsequent assays (or we could just dilute our samples to the same amt of protein) and keep the protein amount added to each assay equivalent for all samples

20

Lowry Assay =
- Hence, according to Beer’s law, the greater the amount of protein, the more...

= used to quantify proteins by measuring their solution absorbance and comparing it to a standard curve
- ...blue-green color is produced, and the greater the amount of light absorbance at 580 nm.

21

Lowry Assay Methods:

1.) Prepare a series of BSA protein standards from 0.8 to 0.025 mg/ml
2.) Prepare your unknowns (from lab 7) according to instructor specifications. (Try diluting S1b and S2 to 1/10 concentration by separately adding 100 μl of the each protein solution to 900 μl of HB. Dilute the resuspended microfibil pellet to 1/10 concentration by adding 100 μl of HB to 900 μl of FRB. Save the remainder of each protein extraction for a future lab).
3.) Prepare 15 ml tubes by labeling them as either BSA concentration, blank or unknown (10 tubes total). Add 1.5 ml of Solution A to each tube then 1.0 ml of either BSA, diluted unknown, or deionized water to the appropriately labeled tube.
4.) Let the solutions sit for 15 minutes at room temperature.
5.) During this time dilute 5 ml of Solution B with 5 ml of H20. It is now at 1.0 N. (There will be an aliquot of diluted Solution B under the fume hood. Only do this step if there is not enough dilute Solution B in the aliquot).
6.) Add 0.4 ml of Solution B to each tube.
7.) Let the solutions sit for 15 min at room temperature. (Precipitate may form at the bottom of the tubes. If so do not resuspend the precipitate or pipette it into the curvette in the next step.)
8.) Read all of the absorbances in the Spec 20 at a wavelength of 580 nm (Curvettes are full at 2.5 ml solution. Do not overfill them. Be sure to calibrate using the blank between measurements.)
9.) Plot a standard curve.
10.) Calculate the protein concentrations for each of the unknowns