GenWay Introduction

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GenWay Introduction
Proteomics Solutions
Unique Proteomic Sample Preparation
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Structures of IgY and IgG
Chicken IgY
Mammalian IgG
Light Chains
Light Chains
CH G2
CH G1
CH G2
MW 150 kDa
CH G Carbohydrate Group
MW 170-190 kDa
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Why Chickens IgYs?
Hen
Rabbit
Distance to Human
-205 Myr
72 Myr
Affinity Maturation
Gene Conversion
Somatic Mutation
Avidity
High
Good
Production
Accumulative
Invasive
Monthly
20 Eggs
50 ml Blood
Ab Quantity
100 mg/egg
10 mg/ml
Maintenance
Low
High
Unit Cost
Low
High
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IgY Less Non-Specific Binding
No Binding to Fc Region
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14 HAP Comprise 96 of the Protein Mass in
Plasma
Transferrin
Fibrinogen
IgG total
IgA
a2-Macroglobulin
IgM
a1-Antitrypsin
Haptoglobin
a1-Acid Glycoprotein
Apolipoproteins A-1 A-II
Albumin
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IgY-Immunoaffinity Separation
IgY-BEADS FEATURES
1. High avidity of binding 2. Specific removal of
the targets 3. Sample size 20-500 ml 4.
Recyclable gt100x 5. Efficacy gt95 6. Minimal
loss of non-targets
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Specific Removal of Targets
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Effect of Separation on Clinical Enzymes
Target Proteins
CRP (mg/dL )
TSH (IU/L)
Samples before Column
0.690.02
1.860.05
Flow-Through from Column
0.510.02
0.870.00
Wash from Column
0.240.01
0.720.03
Eluent from Column
lt0.02
0.180.01
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SuperMix Approach Dig Deeper
Protein Mixture as Antigens for IgY Production
Plasma (P)
IgY12 Column
IgYs
12 HAP-Depleted Fraction (Flow-Through of IgY12
Column F1) (Moderate/Low-Abundant Proteins,
MAP/LAP)
Antigen- Affinity Column
Eluted Fraction (E1) (12 High-Abundant Proteins
HAP)
IgY against 12 HAP-Depleted Fraction (SuperMix)
SuperMix Column
Plasma Directly Applied to IgY-SuperMix Column
Eluted Fraction (E2) (MAP or Immunoreactive
Proteins)
Flow-Through Fraction (F2) (LAP or
Non-Immunoreactive Proteins)
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IgY14 SuperMix Columns
IgY14 Column Contains the IgY antibodies against
the top 14 Highly Abundant Proteins (HAP)
Eluted Fraction (E1) (14 HAP)
Flow-Through Fraction (F1)
IgY-SuperMix Column Contains the IgY antibodies
against the Moderately Abundant Proteins (MAP)
Eluted Fraction (E2) (MAP or Immunoreactive
Proteins)
Flow-Through Fraction (F2) (LAP or
Non-Immunoreactive Proteins)
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Results of SuperMix Column
M P F1 E F2
M MW Marker P Plasma F1 Flow-Through of
IgY-12 E Bound/Eluted Fraction from SuperMix
F2 Flow-Through of IgY-12 IgY-SuperMix 4-20
SDS-PAGE under reducing conditions 4µg Protein
loaded per lane
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IgY-12 vs Tandem IgY-12/SuperMix
Proteoglycan 4 1 ng/mL CD117 4.8
ng/mL E-selectin 6.5 ng/mL Cathepsin D 8.9
ng/mL
LC-MS/MS (triplicate runs)
SCX-LC-MS/MS (25 fractions)
M-CSF1 200 pg/mL P-selectin 120 pg/mL MBP 670
pg/mL PDGFRB 3 ng/mL Leptin 4.7 ng/mL
?2 peptides per protein
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Detected Cytokines/Growth Factors
Analyzed by SCX-LC-MS/MS
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Plant Proteomics Solutions

• Rubisco protein is the biggest obstacle in plant
  proteomics.
• Constitutes 37-55 of the total protein mass in
  green parts of the plant.
• GenWay developed a novel unique Rubisco
  immunodepletion column (multiple use).

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Dow Chemical Published Data
Reference N.A. Cellar, et al., J. Chromatogr. B
(2007), doi10.1016/j.jchromb.2007.11.024
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Dow Chemical Published Data
Arabidopsis and canola RuBisCO bind to the LC
column with little breakthrough into the flow
through fraction. The arabidopsis small chain
fragment appears to be 100 bound because it
could not be detected in the flow through
fraction. Error bars are plotted as standard
deviation with n 3. (B) A gel image showing raw
and flow through (FT) fractions of four different
plant species is shown. Corn and tobacco RuBisCO
was not detected in the raw leaf extracts, so
depletion in these species was not measured
directly.
Reference N.A. Cellar, et al., J. Chromatogr. B
(2007), doi10.1016/j.jchromb.2007.11.024
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Dow Chemical Published Data
RuBisCO elutes at 11 and 12.5 min in the HPLC-UV
chromatogram above. (B) The peak at 3.5 min is an
unknown however, it is depleted by gt98 for all
four species tested. This peak may be a RuBisCO
degradation product or a member of the RuBisCO
interactome.
Reference N.A. Cellar, et al., J. Chromatogr. B
(2007), doi10.1016/j.jchromb.2007.11.024
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Extraction with Urea

• Proteins can be extracted with PBS/0.05 Tween20
  and with Urea/SDS.
• Proteins extracted with SDS/Urea, cannot be
  loaded on the column due to antibody
  incompatibility
• All Rubisco is extracted with PBS/Tween buffer
• In order to extract both hydrophylic and
  hydrophobic proteins one need to extract first
  with PBS/Tween (all the Rubisco in this fraction)
  anf than with Urea/SDS. Use only
  PBS/Tween-extracted proteins for Rubisco removal

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SepproTip Design
SepproTip IgY-12
Membrane filter
500 µl
Stainless steel ring
Membrane filter
IgY Microbeads (Seppro IgY-12)
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SepproTip Process
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SDS-PAGE Analysis of Plasma Protein Fractionation
by SepproTip IgY-12
M Molecular Weight Marker, S Neat plasma, FT
Flow-though fraction from SepproTip IgY-12, E
Eluted fraction from SepproTip IgY-12. 4-20
SDS PAGE under non-reducing condition
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SepproTip Content - Customizable