Isolation, Purification, and Assay of  Wheat Germ Acid Phosphatase            Natalia Manzano            Wilmarie Morales ...
Objectives:• To purify and isolate acid phosphatase  from wheat germ.• Determine protein concentration using  Bicinchonini...
Introduction• Wheat germ is a very small part of a wheat  kernel.• Wheat germ is very high in protein.• It contains around...
Wheat Germ Acid Phosphatase  Catalyzes the hydrolysis of phosphate groups from  macromolecules and smaller molecules that ...
Materials and MethodsMaterials25 g Wheat germ        MnCl2, 1.0 M               Centrifuge, high   BalanceH2O, prechilled ...
Procedure I: Obtaining Fractions                                       Filter wheat germ* All centrifugation was done at  ...
Pellet (buffer)                        Add 33.48 ml of Ammonium Sulfate       Centrifuge                                  ...
Procedure II:   • BCA serves the purpose ofBCA Assay         reacting with complexes                  between copper ions ...
Add Sample+dH2O+BCA
Procedure III: SDS-PAGE• Running Gel:                    • Stacking Gel:  –   1.88 ml dH2O                   –   2.975 ml ...
Get sample obtained                                              from previous purifying                                  ...
Results:      Sample           Absorbance   Blank    Abs-Blank   Conc. (mg/ml)   Vol. of sample added   Dilution factor  F...
Average conc.                  Total proteinSamples          (mg/mL)                               Volumes (mL)           ...
Fraction VI                                                     Fraction V                                                ...
Expected   Obtained
Conclusion• Our results were not what was expected.• We were not able to successfully isolate wheat  germ acid phosphatase...
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...
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  1. 1. Isolation, Purification, and Assay of Wheat Germ Acid Phosphatase Natalia Manzano Wilmarie Morales RISE Program May 10th, 2012
  2. 2. Objectives:• To purify and isolate acid phosphatase from wheat germ.• Determine protein concentration using Bicinchoninic Acid protein assay.
  3. 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. 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. 5. Materials and MethodsMaterials25 g Wheat germ MnCl2, 1.0 M Centrifuge, high BalanceH2O, prechilled to 4˚C Sodium acetate speed buffer, 1.0M (pH 5.7) Ice bucket SpectrophotometCheesecloth (NH4)2SO4, saturated er, visible (pH 5.5) Magnetic stirrer MicroplateIce, crushed Pasteur Pipets Waterbaths (30˚C and 70˚C)BCA Kit Gloves, disposableBSA standard, 1.0 Weighing traysmg/mlMethods Bicinchoninic Acid protein assay (BCA) SDS PAGE
  6. 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. 7. Pellet (buffer) Add 33.48 ml of Ammonium Sulfate Centrifuge (Saturated 35%) Supernatant 92 ml CentrifugeDiscard 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 FractionDiscard Pellet VI
  8. 8. Procedure II: • BCA serves the purpose ofBCA 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. 9. Add Sample+dH2O+BCA
  10. 10. 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
  11. 11. Get sample obtained from previous purifying technique Load BufferSet up gel, removecomb Load Sample (3:1 sample:buffer) Run GelStain with Coomassie andStudy Gel Procedure III: SDS
  12. 12. 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 10Fraction I X/100-L 0.13 0.094 0.036 37.930 2.3 100Fraction 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 100Fraction 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 100Fraction 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 10Fraction 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 10 Fraction V X/10-H 0.673 0.094 0.579 12.201 11.5 10Fraction V X/100-L 0.151 0.094 0.057 60.055 2.3 100Fraction 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 10Fraction 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
  13. 13. Average conc. Total proteinSamples (mg/mL) Volumes (mL) (mg)Fraction I 30.39 65 1974.62Fraction II 64.16 62 3978.18Fraction III 5.92 25 147.90Fraction IV 7.56 21 158.86Fraction V 34.21 136 4652.61Fraction VI 33.94 78 2647.45
  14. 14. Fraction VI Fraction V Fraction V Fraction III Fraction II Fraction I 250- 150- 100- 75- 50- 37- 25- 15-PAGESDS-
  15. 15. Expected Obtained
  16. 16. 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, ).
  17. 17. Acknowledgements • Vibha Bansal PhD • Edmarie Martinez • Vivian Rodriguez Cruz• Alexandra Rosado Burgos
  18. 18. 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]

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