1. +
Isolation, Purification,
and Assay of Wheat
Germ Acid Phosphatase
Natalia Manzano
Wilmarie Morales
RISE Program
May 10th, 2012

2. +
Introduction
 Wheat germ is a very small part of a wheat kernel.
 Wheat germ is very high in protein.
 It contains around 28% protein and has more protein than can be
found in most meat products.
 The human body needs protein in order to repair tissue damage
and to help minerals and nutrients reach our cells.
 It contains more potassium and iron than any other food source.
 Also has calcium, zinc, magnesium, and vitamins A, B1, B3 and E.
 Vitamins B1 and 3 are very important to maintain energy levels
and maintain healthy muscles and organs. Vitamin E is a very
important antioxidant, prevents heart disease, and is needed to
strengthen the body’s immune system.

3. +
Acid Phosphatase
 Acid phosphatase is a type of enzyme used to free attached
phosphate groups from other molecules during digestion.
 It is basically a phosphomonoesterase, a phosphatase that
acts on monoesters.
 It is stored in lysosomes and functions when these fuse
with endosomes.
 Phosphatase enzymes are also used by soil microorganisms
to access organically bound phosphate nutrients.
 Some plant roots, exude carboxylates that perform acid
phosphatase activity, helping to mobilise phosphorus in
nutrient-deficient soils.

4. + Wheat Germ Acid Phosphatase
• Acid phosphatase ocurres in plants and animals,
and has a pH optimum below 7.
• Catalyzes the hydrolysis of phosphate groups from
macromolecules and smaller molecules that are
stored in the wheat seed.

5. http://www.google.com.pr/imgres?imgurl=http://nutritioncheckup.com/blog/wp-
content/uploads/2010/10/wheat-germ.jpg&imgrefurl=http://nutritioncheckup.com/blog/?p
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+ • The growing wheat embryo uses the freed
phosphate in germination and growth.

6. +
Objectives
 To
purify and isolate acid phosphatase from
wheat germ.
 Determine protein concentration using
Bicinchoninic Acid protein assay.

7. +
Metodology
• First we isolated acid phosphatase by suspending
25g of wheat germ in 100 mL prechilled dH20.
Filtrated with cheesecloth approximately 70 mL of
solution. Centrifuge the filtrate at 4˚C for 20
minutes at 2,800 x g. Collected 65 mL of the
supernatant denoted as Fraction I.
• Then purified the acid phosphatase, we transfered
Fraction I to a 150 mL beaker, and added 0.396g of
1.0M MnCl2 to 1.0 ml of dH2O. And added 1.26 ml
of 1.0 M MnCl2 to Fraction I.
• Collect 62 mL of the other centrifuged
supernatant from Fraction I denoted as Fraction
II.

8. +
• Suspended the pellet with 4.8 mL of 0.05 M of
Solution Acetic Acid and 45.2 mL of 0.05 M
Sodium Acetate. The supernatant obtained oof 25
mL was denoted as Fraction III.
• Transferred the remainder of Fraction II to a 400
ml beaker placed in an ice bath. Added 33.48 ml
of (NH4)2SO4 to Fraction II. Centrifuged Fraction II
and obtained 92 mL of the supernatant.

9. +
• Suspend the centrifuged pellets and added 20 ml of 0.05 M
sodium acetate buffer, pH 5.67. The 21 mL of supernatant is
denoted as Fraction IV.
• Add 46.92 mL (NH4)2SO4 to Fraction II.
• WILMA YOU DID THIS????
• Collected 136 mL of supernatant. This is denoted as
Fraction V
• Suspended the pellet obtained in 20 ml of cold, dH2O.
Centrifuge the solution to remove undissolved protein.
The78 mL supernatant is denoted as Fraction VI. Record
the volume of Fraction VI.

10. + Materials and Methods
Materials
25 g Wheat germ MnCl2, 1.0 M
H2O, prechilled to Sodium acetate
4˚C buffer, 1.0M (pH
5.7)
Cheesecloth (NH4)2SO4, saturated
(pH 5.5) Methods
Ice, crushed Pasteur Pipets
BCA Kit Gloves, disposable Bicinchoninic Acid protein assay
BSA standard, 1.0 Weighing trays (BCA)
mg/ml
SDS PAGE
Centrifuge, high Balance
speed
Ice bucket Spectrophotometer
, visible
Magnetic stirrer Microplate
Waterbaths (30˚C
and 70˚C)

11. +
Procedure I: Obtaining Fractions
* All centrifugation was done Filter wheat germ
at 2800Xg with cheese cloth
at 4° for 20min
Centrifuge filtrate (70ml)
Discard Pellet Fraction I
Pellet Add 1.26 ml of MnCl2 1.0M
Discard Pellet Add Sodium Acetate Centrifuge
Centrifuge Supernatant 62 ml
Supernatant 25 ml Fraction II
Fraction III

12. Pellet Add 33.48 ml of Ammonium Sulfate
(buffer) (Saturated 35%)
Centrifuge Supernatant 92 ml
Centrifuge
Discard Pellet
Add 46.92 ml of
Supernatant Ammonium Sulfate
21 ml (Saturated 57%)
Centrifuge
Supernatant 136 ml
Fraction IV
Pellet FractionV
Suspend in dH2O
Centrifuge
Fraction
Discard Pellet VI

13. +
 BCA serves the purpose of
reacting with complexes
between copper ions and
peptide bonds to produce
Procedure II: a purple end product.
BCA Assay  Estimate visually or
measure with a standard
spectrophotometer or
plate reader (562nm).

14. +
Add
Sample+
dH2O+BCA

15. +
Conc.
Of
Abs Vol. of
working Blan Abs- Conc.
orba sample
std k Blank (mg/ml)
nce added
(mg/mL Stock
) used
0.16 0.09
1X 0.068 0.200 2.3
1 2 4
0.42 0.09
1X 0.330 0.600 6.9
4 4
0.54 0.09
1X 0.446 1.000 11.5
0 4
1X/1 0.10 0.09
0.007 0.020 2.3
0.1 0 1 4
1X/1 0.12 0.09
0.032 0.060 6.9
0 6 4
1X/1 0.12 0.09
0.034 0.100 11.5
0 8 4
1X/1 0.07 0.09
0.000 0.002 2.3
0.01 00 8 4
1X/1 0.08 0.09
0.000 0.006 6.9
00 3 4 Blank Concentration Curve
1X/1 0.09 0.09
0.000 0.010 11.5
00 4 4

16. +
Procedure III: SDS-PAGE
 Running Gel:  Stacking Gel:
 1.88 ml dH2O  2.975 ml dH2O
 1.67 ml separating buffer  1.25 ml separating buffer
 2.22 ml Acrylamide  0.662 ml Acrylamide
 50.0 µl 20% SDS  225.0 µl 20% SDS
 100 µl 10% Glycerol  25 µl 10% Glycerol
 1.67 µl TEMED  6.25 µl TEMED
 50 µl APS(100 mg/ml)  25 µl APS

17. +
Procedure III: SDS
Get your sample
obtained from previous
purifying technique
(Purification)
Set up gel, remove
Load Buffer
comb
Stain and look at with Load Sample (3:1
Run Gel sample:buffer)
UV light

18. Results:
Sample Absorbance Blank Abs-Blank Conc. (mg/ml) Vol. of sample added Dilution factor
Fraction I 1X-L 2.511 0.094 2.417 25.466 2.3 1
Fraction I 1X-H 3.029 0.094 2.935 6.185 11.5 1
Fraction I X/10-L 0.305 0.094 0.211 22.231 2.3 10
Fraction I X/10-H 1.171 0.094 1.077 22.695 11.5 10
Fraction I X/100-L 0.13 0.094 0.036 37.930 2.3 100
Fraction I X/100-H 0.241 0.094 0.147 30.976 11.5 100
Fraction II 1X-L 3.029 0.094 2.935 30.923 2.3 1
Fraction II 1X-H 3.029 0.094 2.935 6.185 11.5 1
Fraction II X/10-L 0.385 0.094 0.291 30.660 2.3 10
Fraction II X/10-H 1.636 0.094 1.542 32.493 11.5 10
Fraction II X/100-L 0.164 0.094 0.07 73.752 2.3 100
Fraction II X/100-H 0.512 0.094 0.418 88.081 11.5 100
Fraction III 1X-L 0.524 0.094 0.43 4.530 2.3 1
Fraction III 1X-H 2.035 0.094 1.941 4.090 11.5 1
Fraction III X/10-L 0.143 0.094 0.049 5.163 2.3 10
Fraction III X/10-H 0.317 0.094 0.223 4.699 11.5 10
Fraction III X/100-L 0.094 0.094 0 0.000 2.3 100
Fraction III X/100-H 0.138 0.094 0.044 9.272 11.5 100
Fraction IV 1X-L 0.536 0.094 0.442 4.657 2.3 1
Fraction IV 1X-H 1.541 0.094 1.447 3.049 11.5 1
Fraction IV X/10-L 0.149 0.094 0.055 5.795 2.3 10
Fraction IV X/10-H 0.303 0.094 0.209 4.404 11.5 10
Fraction IV X/100-L 0.102 0.094 0.008 8.429 2.3 100
Fraction IV X/100-H 0.163 0.094 0.069 14.540 11.5 100
Fraction V 1X-L 2.373 0.094 2.279 24.012 2.3 1
Fraction V 1X-H 3.029 0.094 2.935 6.185 11.5 1
Fraction V X/10-L 0.179 0.094 0.085 8.956 2.3 10
Fraction V X/10-H 0.673 0.094 0.579 12.201 11.5 10
Fraction V X/100-L 0.151 0.094 0.057 60.055 2.3 100
Fraction V X/100-H 0.358 0.094 0.264 55.630 11.5 100
Fraction VI 1X-L 1.443 0.094 1.349 14.213 2.3 1
Fraction VI 1X-H 3.029 0.094 2.935 6.185 11.5 1
Fraction VI X/10-L 0.212 0.094 0.118 12.432 2.3 10
Fraction VI X/10-H 0.657 0.094 0.563 11.864 11.5 10
Fraction VI X/100-L 0.152 0.094 0.058 61.109 2.3 100
Fraction VI X/100-H 0.333 0.094 0.239 50.362 11.5 100

19. Total
Average conc.
Samples (mg/mL)
Volumes (mL) protein
(mg)
Fraction I 30.39 65 1974.62
Fraction II 64.16 62 3978.18
Fraction III 5.92 25 147.90
Fraction IV 7.56 21 158.86
Fraction V 34.21 136 4652.61
Fraction VI 33.94 78 2647.45

20. +
Expected Concentration Curve Fraction Concentration Curve

21. +
Expected Trend Experimental Trend

22. Fraction VI
Fraction V
Fraction V
Fraction III
Fraction II
Fraction I
250-
150-
100-
75-
50-
37-
25-
15-
PAGE
SDS-
+

23. +
Gel Results
Expected Obtained

24. +
Conclusion
 Our results were not what was expected.
 We were not able to successfully isolate wheat germ acid
phosphatase.
 This could be accountable to human error (bad pipetting,
error in making solutions, etc.).

25. +
Acknowledgements
 Vibha Bansal PhD
 Edmarie Martinez
 Vivian Rodriguez Cruz
 Alexandra Rosado Burgos

26. +
References
 Stoscheck ,2000. CM. Quantitation of Protein. Methods in
Enzymology, 50-69.
 Yasuaki Kawarasaki, Hideo Nakano, Tsuneo Yamane, 1999.
Purification and some properties of wheat germ acidphosphatases.
Elsvier
[http://www.sciencedirect.com/science/article/pii/016894529604
4779]
 Ke-Xue Zhu,Hui-Ming Zhou, and Hai-Feng Qian, 2008. Proteins
Extracted from Defatted Wheat Germ: Nutritional and Structural
Properties
[http://cerealchemistry.aaccnet.org/doi/abs/10.1094/CC-83-0069
]