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Isolation, Purification, and Assay of Wheat Germ Acid Phosphatase

Natalia ManzanoWilmarie Morales

RISE ProgramMay 10th, 2012

Objectives:

• To purify and isolate acid phosphatase from wheat germ.

• Determine protein concentration using Bicinchoninic Acid protein assay.

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.

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.

Materials and Methods

Materials

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

Methods

Centrifuge, high speed

Balance

Ice bucket Spectrophotometer, visible

Magnetic stirrer MicroplateWaterbaths (30˚C and 70˚C)

Bicinchoninic Acid protein assay (BCA)

SDS PAGE

Procedure I: Obtaining FractionsFilter wheat germ with cheese cloth

Centrifuge filtrate (70ml)

Fraction IDiscard Pellet

Add 1.26 ml of MnCl2 1.0MCentrifuge

Supernatant 62 ml

* All centrifugation was done at 2800Xg

at 4° for 20min

Fraction II

PelletAdd Sodium Acetate

CentrifugeDiscard Pellet

Supernatant 25 ml

Fraction III

Add 33.48 ml of Ammonium Sulfate (Saturated 35%)

Supernatant 92 mlCentrifuge

Pellet (buffer)Centrifuge

Discard Pellet

Supernatant21 ml

Fraction IV

Add 46.92 ml of Ammonium Sulfate

(Saturated 57%)Centrifuge

Supernatant 136 ml

Fraction VPellet Suspend in dH2O

Centrifuge

Discard Pellet

Fraction VI

Procedure II: BCA Assay

• BCA serves the purpose of 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).

Add Sample+ dH2O+BCA

Procedure III: SDS-PAGE

• Running Gel:

– 1.88 ml dH2O

– 1.67 ml separating buffer

– 2.22 ml Acrylamide

– 50.0 µl 20% SDS

– 100 µl 10% Glycerol

– 1.67 µl TEMED

– 50 µl APS(100 mg/ml)

• Stacking Gel:

– 2.975 ml dH2O

– 1.25 ml separating buffer

– 0.662 ml Acrylamide

– 225.0 µl 20% SDS

– 25 µl 10% Glycerol

– 6.25 µl TEMED

– 25 µl APS

Procedure III: SDS

Get sample obtained from previous purifying technique

Set up gel, remove comb

Load Buffer

Load Sample (3:1 sample:buffer)

Run Gel

Stain with Coomassie and Study Gel

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 1Fraction 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 10Fraction 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 10Fraction 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 100Fraction 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 10Fraction 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 100Fraction VI X/100-H 0.333 0.094 0.239 50.362 11.5 100

SamplesAverage conc.

(mg/mL)Volumes (mL)

Total 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

250-

150-

100-

75-

50-

37-

25-

15-

Fraction

VI

Fraction

V

Fraction

V

Fraction

III

Fraction

II

Fraction

I

SDS-PAGE

Expected Obtained

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, ).

Acknowledgements

• Vibha Bansal PhD

• Edmarie Martinez

• Vivian Rodriguez Cruz

• Alexandra Rosado Burgos

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/0168945296044779]