1. Protocol…Isolation Purificationand Assay of Wheat Germ Acid Phosphatase… May 3-4, 2012
Isolation, Purification, and Assay of Wheat Germ Acid Phosphatase
I. Materials
Reagent/ Supplies
25 g Wheat germ MnCl2, 1.0 M
H2O, prechilled to 4˚C Sodium acetate buffer, 1.0M (pH 5.7)
Cheesecloth (NH4)2SO4, saturated (pH 5.5)
Ice, crushed Pasteur Pipets
BCA Kit Gloves, disposable
BSA standard, 1.0 mg/ml Weighing trays
II. Equipment/ Apparatus
Centrifuge, high speed Balance
Ice bucket Spectrophotometer, visible
Magnetic stirrer Microplate
Waterbaths (30˚C and 70˚C)
III. Isolation of Acid Phosphatase
1. Suspend 25 g of wheat germ in 100 ml of prechilled (4˚C) dH2O. Let the mixture
stand for 30 min with magentic stirrer.
2. Form a sac with two layers of cheesecloth and pass the suspension through it. Squeeze
the residue as dry as possible. The material passed through the cheesecloth is the
filtrate. Discard the dry residue inside the sac.
Filtrate volume: ~70 mL
3. Centrifuge the filtrate in the cold (4˚C) for 20 minutes at 2,800 x g.
4. Decant the supernatant into a graduated cylinder by pouring the liquid carefully over
the side containing the pellet. This minimizes the chance of accidentally loosening the
pellet and removing all or some of it with the supernatant.
5. Discard the pellets and collect the spernatant; record its volume. The supernatant is
denoted as Fraction I. This type of fraction is known as the crude extract in protein
and enzyme isolations. It represents the first fraction after removal of intact cells and
cell debris.
Fraction I volume: 65 ml
6. Remove and freeze a 1.0 ml aliquot of Fraction I for later assay of protein
concentration and enzyme activity. Remember to include this aliquot in summarizing
the results for Fraction I in your final purification table. Do likewise for the other
aliquots saved later.
IV. Purification of Acid Phosphatase
1. Place a plastic bucket, filled with ice, on a magnetic stirrer. Insert a 150 ml beaker
into the ice.
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2. Protocol…Isolation Purificationand Assay of Wheat Germ Acid Phosphatase… May 3-4, 2012
2. Transfer Fraction I to the beaker and add 2.0 ml of 1.0 M MnCl2 for every 100 ml
Fraction I being processed. Stir gently during the additon of the MnCl2.
• Calculation to prepare 1.0 M MnCl2 :
1 m M l  9. g n l  1 
. o n 2 17 M 2  L
0 l C 9 C
•   (. l 0 6 M 2
0 ) 3
 3 2 m = . 9g n l
C
1L m n 2 0 l
1 oM l 1 m
l C 
• Add 0.396g of 1.0M MnCl2 to 1.0 ml of dH2O. Let dissolve using
magnetic stirrer. Make up the volume with dH2O to 2.0 ml.
• In order to determine the amount of 1.0M MnCl2 that needed to be added
100m 63m
l l
=
we calculated this proportion: 2.0m l x
x =1.26m M l2
l nC
• So, we added: 1.26 ml of 1.0 M MnCl2 to Fraction I.
3. Repeat the procedure of steps III-3 and III-4. Collect the supernatant which is denoted
as Fraction II. Record the volume.
Fraction II volume: 62 ml
4. Suspend the pellets in 25 ml of 0.05 M sodium acetate buffer (pH 5.7) by means of a
stirring rod. When the suspension appears reasonably uniform, remove undissolved
protein by a 20 minute centrifugation at 2,800 x g in the cold (4˚C). The pellet
obtained at this point may be discarded. The supernatant (record its volume) is
denoted as Fraction III and is saved in its entirety. Remove and freeze a 1.0 ml
aliquot, then freeze the remainder of the fraction separately.
Fraction III volume: 25 ml
• Prepare 0.05 M Sodium Acetate Buffer (pH 5.7)
• Add 4.8 mL of 0.05 M of Solution Acetic Acid + 45.2 mL of 0.05
M Sodium Acetate. Make up the volume to 100 ml, pH 5.67
5. While little activity is expected in Fraction III, as well as in some of the other fractions
to be obtained later, it is advisable to retain all of the fractions obtained during enzyme
purification until they are shown to contain negligible activity. This practice allows
one to pinpoint those steps in the purification at which problems may arise.
6. Repeat the procedure of Step V-6 for Fraction II. Transfer the remainder of Fraction II
to a 400 ml beaker placed in an icebath (see step IV-1).
7. Add slowly, and with gently stirring, cold, saturated ammonium sulfate to Fraction II.
Add 54 ml of ammonium sulfate for every 100 ml of Fraction II being processed. This
brings the solution to 35% saturation in ammonium sulfate. The additon of
ammonium sulfate should be done slowly, over a period of 5-10 minures, to avoid
denaturation of proteins. Such denaturation is indicated by the formation of an off-
white foam at the surface of the solution.
• To Prepare (NH4)2SO4, saturated (pH 5.5) at 4˚C.
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3. Protocol…Isolation Purificationand Assay of Wheat Germ Acid Phosphatase… May 3-4, 2012
• Calculated the amount of Ammonium sulfate using this proportion to
1 0g
56 x
=
obtain a saturated solution: 2 0m 1
00 l 20m l
x=9 .6g (N 4)2S 4
3 H O
• Add 93.6 g of (NH4)2SO4 to 120 mL of dH2O. Store beaker in 4˚C. Let
salt dissolve with magnetic stirrer.
• Proportion calculated in order to determine how much ammonium
10 6
0 2
=
sulfate needed to be added to Fraction II: 5 4 x
x=3 .4 m (N 4)2S 4
3 8 l H O
• Add 33.48 ml of (NH4)2SO4 to Fraction II.
8. Continue stirring for 10-15 minutes after all of the ammonium sulfate has been added.
9. Repeat the procedure of Steps V-3 and V-4.
10. Determine the volume of the supernatant and transfer it back unto the 400 ml beaker in
the icebath for a second fractionation with ammonium sulfate.
Volume of supernatant: 92 ml
11. Suspend the pellets from step IV-9 by the procedure of Step IV-4 (added 20 ml of 0.05
M sodium acetate buffer, pH 5.67). The pellet obtained at this point may be discarded.
The supernatant is denoted as Fraction IV. Remove and freeze a 1.0 ml aliquot, then
freeze the remainder of the fraction seperately.
Fraction IV volume: 21 ml
12. To the solution of step IV-10 add slowly, with gentle stirring, 51 ml of cold, saturated
ammonium sulfate for every 100 ml of solution being processed. This brings the
solution to 57% saturation in ammonium sulfate.
• Calculated proportion to determine how much saturated ammonium
10 9
0 2
=
sulfate needed to be added to Fraction II: 51 x
x=4 .9 m (N 4)2S 4
6 2 l H O
• Add 4 2 lN 2O Fraction II.
6 mH 4to
.
9 ( 4S
)
13. Prepare a 70˚C waterbath and an ice-water-bath which can accommodate the beaker
from step IV-12.
14. Transfer the beaker from step IV-12 to the 70˚C waterbath and stir gently with a
thermometer. Allow the solution to warm to 60˚C and maintain it at that temperature
for exactly 2 minutes. After this heat treatment, plunge the beaker quickly into the ice-
water bath. Stir the solution continuously and carefully with the thermometer until its
temperature has dropped to 8˚C.
15. Repeat the procedure of steps III-3 and III-4.
16. Collect the supernatant in its entirety and record its volume. This is denoted as
Fraction V. Remove and freeze a 1.0 ml aliquot, then freeze the remainder of the
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4. Protocol…Isolation Purificationand Assay of Wheat Germ Acid Phosphatase… May 3-4, 2012
fraction separately.
Fraction V volume: 136 ml
17. Suspend the pellet obtained in step V-15 in 20 ml of cold, dH2O. After the pellet has
been evenly suspended, centrifuge the solution for 20 minutes at 2,800 x g in the cold
(4˚C) to remove undissolved protein. The pellet obtained at this point may be
discarded. The supernatant is denoted as Fraction VI. Record the volme of Fraction
VI. If desired, the purification may be interrupted at this point, and the preparation
may be stored frozen at -20˚C for several weeks without significant loss of activity.
Fraction VI volume: 78 ml
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