BIOLOGY 371

INDEPENDENT RESEARCH

WEEK 2

Cellular Fractionation and Centrifugation

1. Reversible denaturation of proteins using ammonium sulphate.
2. Centrifugation.
3. "Salting out" profile for Isocitrate dehydrogenase.

Ammonium Sulphate Fractionation of IDH

[ All procedures are to be performed at 4° C.]
1. Homogenize 100 - 200 gm of tissue in 3 volumes of cold homogenization buffer. [Plant tissues may need to be frozen at -70° C to rupture cell walls.]
2. Pour homogenate through 2 layers of cheese cloth.
3. Centrifuge filtrate at 17,000 g for 15 minutes.
4. Save the supernatant, recording its volume. This is the crude homogenate.
5. Place the supernatant in a beaker, add a magnetic stir-bar, and make the solution 35% saturated with ammonium sulfate. Add the appropriate amount of ammonium sulfate [Table 1] slowly while stirring. Stir for 15 minutes.
5. Centrifuge the 35% preparation at 17,000 g for 15 minutes.
6. Re-suspend the pellet in a minimal volume of homogenization buffer. Label this 35% P and store on ice.
7. Place the 35% saturated supernatant in a beaker, add enough ammonium sulfate [Table 1] to make the solution 45% saturated and stir for 15 minutes.
8. Centrifuge the 45% preparation at 17,000 g for 15 minutes.
9. Re-suspend the pellet in a minimal volume of homogenization buffer. Label this 45% P and store on ice.
10. Place the 45% saturated supernatant in a beaker, add enough ammonium sulfate to make the solution 55% saturated and stir for 15 minutes.
11. Continue this procedure to produce a 55% P, a 65% P, a 75% P and a 75%
supernatant.
12. Perform triplicate IDH assays for each of these fractions and record your data as follows:

Cellular Fractionation of Tissue

Fraction	Volume [mL]	Activity*
Crude Homogenate
35% Pellet
45% Pellet
55% Pellet
65% Pellet
75% Pellet
75% Supernatant

*These data may be "relative activity".

10. Now you must decide on the minimum and maximum levels of ammonium sulfate saturation to use the first step in purifying isocitrate dehydrogenase from your experimental material. For example, if there are high levels of IDH activity in the 45%, 55% and 65% pellets but none in the 35% and 75% pellets, you may chose the following procedure for future experiments:

i. Make your crude homogenate 45% saturated with ammonium sulfate.
ii. Discard the pellet.
iii. Make a 65% pellet and discard the supernatant.

Ammonium Sulfate Table

This table indicates the correct amount of solid ammonium sulfate [at 25° C] to be added to one litre of solution to produce a desired change in the percent saturation of ammonium sulfate. Saturated ammonium sulfate at 25° C is 4.1 M and requires 767 grams of salt per litre.

Percent Saturation - Final

Initial	20	25	30	35	40	45	50	55	60	65	70	75
0	114	144	176	209	243	277	313	351	390	430	472	516
20		29	59	91	123	155	189	225	262	300	340	382
25			30	61	93	125	158	193	230	267	307	348
30				30	62	94	127	162	198	235	273	314
35					31	63	94	129	164	200	238	278
40						31	63	97	132	168	205	245
45							32	65	99	134	171	210
50								33	66	101	137	176
55									33	67	103	141
60										34	69	105
65											34	70
70												35

The Effects of Salt on Proteins

[Summarized from Suelter]

Salts affect the electrostatic and nonpolar properties of proteins in a reversible manner. At concentrations above 0.2M, salts not only neutralize the electrostatic forces on the protein surface but also affect the three dimensional structure of proteins, making them less soluble. Separating proteins from a crude extract by "salting out" using ammonium sulfate is a convenient purification step [see Week 3, Table 1].

Return to Schedule

© Copyright 2000 Department of Biology, Davidson College, Davidson, NC 28036
Send comments, questions, and suggestions to: jowilliamson@davidson.edu