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

2. Objectives:
• To purify and isolate acid phosphatase
from wheat germ.
• Determine protein concentration using
Bicinchoninic Acid protein assay.

3. 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.

4. Wheat Germ Acid Phosphatase
Catalyzes the hydrolysis of phosphate groups from
macromolecules and smaller molecules that are stored in the
wheat seed. The growing wheat embryo uses the freed
phosphate in germination and growth.

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

6. Procedure I: Obtaining Fractions
Filter wheat germ
* All centrifugation was done at with cheese cloth
2800Xg
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

7. Pellet (buffer) Add 33.48 ml of Ammonium Sulfate
Centrifuge (Saturated 35%)
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 Fraction V
Suspend in dH2O
Centrifuge
Fraction
Discard Pellet VI

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

9. Add Sample+
dH2O+BCA

11. 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

12. Get sample obtained
from previous purifying
technique
Load Buffer
Set up gel, remove
comb
Load Sample (3:1
sample:buffer)
Run Gel
Stain with Coomassie and
Study Gel
Procedure III: SDS

13. 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

14. Average conc. Total protein
Samples (mg/mL)
Volumes (mL)
(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

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

18. Expected Obtained

19. 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, ).

20. Acknowledgements
• Vibha Bansal PhD
• Edmarie Martinez
• Vivian Rodriguez Cruz
• Alexandra Rosado Burgos

21. 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/articl
e/pii/0168945296044779]