The document describes experiments to isolate and characterize acid phosphatase from wheat germ. It outlines the objectives, introduction, materials and methods used which included fractionating the wheat germ extract through ammonium sulfate precipitation and measuring protein concentration via BCA assay. The results showed the highest protein concentrations in Fractions II and V but SDS-PAGE analysis did not show clear bands, suggesting acid phosphatase was not successfully isolated. Possible sources of error are acknowledged.
Particle Size Analysis for Homogenization Process Development HORIBA Particle
Emulsions and suspensions are commonly used in pharmaceutical, chemical and consumer products. The pharmaceutical industry, in particular, uses emulsions and suspensions to increase drug efficacy by controlling their particle size and size distribution. Among various available preparation methods, high-pressure homogenization is one of the widely employed processes in the field. This webinar discusses ways to develop a robust homogenization process for making pharmaceutical emulsions by evaluating droplet size distribution.
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Particle Size Analysis for Pharmaceutical Homogenization Process DevelopmentHORIBA Particle
Guest speaker Dr. Daniel Huang of Novartis joins HORIBA Scientific (http://www.horiba.com/particle) to speak about homogenization process development.
High-pressure homogenization has been used to prepare emulsions in a broad range of industries. Particle size distribution and emulsion stability are two key properties of the system essential to a successful downstream application. In this study, Daniel and his colleagues have investigated the effects of process conditions on droplet size and coalescence rate of the emulsion systems.
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Particle Size Analysis for Homogenization Process Development HORIBA Particle
Emulsions and suspensions are commonly used in pharmaceutical, chemical and consumer products. The pharmaceutical industry, in particular, uses emulsions and suspensions to increase drug efficacy by controlling their particle size and size distribution. Among various available preparation methods, high-pressure homogenization is one of the widely employed processes in the field. This webinar discusses ways to develop a robust homogenization process for making pharmaceutical emulsions by evaluating droplet size distribution.
View recorded webinars:
http://bit.ly/particlewebinars
Particle Size Analysis for Pharmaceutical Homogenization Process DevelopmentHORIBA Particle
Guest speaker Dr. Daniel Huang of Novartis joins HORIBA Scientific (http://www.horiba.com/particle) to speak about homogenization process development.
High-pressure homogenization has been used to prepare emulsions in a broad range of industries. Particle size distribution and emulsion stability are two key properties of the system essential to a successful downstream application. In this study, Daniel and his colleagues have investigated the effects of process conditions on droplet size and coalescence rate of the emulsion systems.
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Spotlight on Analytical Applications Complete e-Zine Vol. 1PerkinElmer, Inc.
This document provides key analytical applications to help laboratories address the pressing concerns of the changing global landscape. Specifically, Volume 1 includes applications for Children's Product Safety, Environmental, Food & Beverage and Semiconductor.
Compatibility of DMSO spotting buffers with Nexterion Slide E Epoxy-coated Mi...SCHOTT
Compatibility of DMSO spotting buffers with SCHOTT Nexterion Epoxy-silane coated microarray slides. Effect of using different spotting pins and DMSO concentrations.
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We are a products, solutions and services wholesale supplier in water treatment. We provide only businesses companies such as distributors, retailers, manufacturers and assemblers.A catalog including carefully selected products, from the most prestigious brands in the Water Treatment market, operative website, personalized services, precise and efficient logistics, flexible organization, but also the basic importance that is attributed to human factors and relationships with partners, make of Sinergroup Srl a reference for many companies of the market. A continuous and consistent effort, the ability to anticipate the evolution of the market demand and orientation to the understanding and satisfaction of our customers has always characterizes us in our work.
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Modern Particle Characterization Techniques Series: Laser DiffractionHORIBA Particle
This part two of the webinar series will introduce participants to basic experimental considerations when choosing laser diffraction for particle size analysis. The presentation will explain what makes laser diffraction a “modern technique.” Both wet and dry case studies will be shown along with brief demonstration videos.
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Spotlight on Analytical Applications Complete e-Zine Vol. 1PerkinElmer, Inc.
This document provides key analytical applications to help laboratories address the pressing concerns of the changing global landscape. Specifically, Volume 1 includes applications for Children's Product Safety, Environmental, Food & Beverage and Semiconductor.
Compatibility of DMSO spotting buffers with Nexterion Slide E Epoxy-coated Mi...SCHOTT
Compatibility of DMSO spotting buffers with SCHOTT Nexterion Epoxy-silane coated microarray slides. Effect of using different spotting pins and DMSO concentrations.
activated, big, carbon, carbon block, cartridges, ceramic candle, demineralization, double layers, drinking water at home, filter, gac, granular activated carbon, melt, pleated, polyester, polypropylene, resin, stainless steel, washable, wound sediment
We are a products, solutions and services wholesale supplier in water treatment. We provide only businesses companies such as distributors, retailers, manufacturers and assemblers.A catalog including carefully selected products, from the most prestigious brands in the Water Treatment market, operative website, personalized services, precise and efficient logistics, flexible organization, but also the basic importance that is attributed to human factors and relationships with partners, make of Sinergroup Srl a reference for many companies of the market. A continuous and consistent effort, the ability to anticipate the evolution of the market demand and orientation to the understanding and satisfaction of our customers has always characterizes us in our work.
Born with the objective view in the exclusive distribution: Continue to be "Leader of the Brand Leader ", with activities and services of value. A choice of specialization that in recent years has allowed us to develop a unique knowledge and experience in the market. The thorough water treatment market understanding and high commercial performance, make us able to offer to our customers many products and services more personalized and appropriate to the individual end user. Our mission is to become your best business Partner!
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We are a products, solutions and services wholesale supplier in water treatment. We provide only businesses companies such as distributors, retailers, manufacturers and assemblers.A catalog including carefully selected products, from the most prestigious brands in the Water Treatment market, operative website, personalized services, precise and efficient logistics, flexible organization, but also the basic importance that is attributed to human factors and relationships with partners, make of Sinergroup Srl a reference for many companies of the market. A continuous and consistent effort, the ability to anticipate the evolution of the market demand and orientation to the understanding and satisfaction of our customers has always characterizes us in our work.
Born with the objective view in the exclusive distribution: Continue to be "Leader of the Brand Leader ", with activities and services of value. A choice of specialization that in recent years has allowed us to develop a unique knowledge and experience in the market. The thorough water treatment market understanding and high commercial performance, make us able to offer to our customers many products and services more personalized and appropriate to the individual end user. Our mission is to become your best business Partner!
Modern Particle Characterization Techniques Series: Laser DiffractionHORIBA Particle
This part two of the webinar series will introduce participants to basic experimental considerations when choosing laser diffraction for particle size analysis. The presentation will explain what makes laser diffraction a “modern technique.” Both wet and dry case studies will be shown along with brief demonstration videos.
In this webinar, you will learn:
- Method development
- Choosing an appropriate refractive index
- Understanding the analysis results
View recorded webinars:
http://bit.ly/particlewebinars
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Detailed hydrocarbon (DHA) analysis of automotive gasoline has become a routine gas chromatographic method. The Canadian standard CAN/CGSB-3.0 requires cryogenic temperature programing to provide better separation of early-eluting components than conventional non-cryogenic DHA measurements. Determination of aromatics in the presence of methanol and ethanol is also an important requirement for bio-ethanol; method ASTM D-5580 is the standard method for this analysis.
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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).
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
15.
16.
17. Fraction VI
Fraction V
Fraction V
Fraction III
Fraction II
Fraction I
250-
150-
100-
75-
50-
37-
25-
15-
PAGE
SDS-
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, ).