Download Capillary Electrophoresis of Proteins

Capillary Electrophoresis
of Proteins
SDS Capillary Gel Electrophoresis
SDS-CGE
Outline
• CE-SDS Gel Analysis
– Description of Technique
– Method Development Tips
• PA800 plus kits
– SDS-MW
– IgG Purity & Heterogeneity
CE-SDS Gel Analysis
• Capillary Gel Electrophoresis
– Molecular sieving
• CE separations are based on analytes’ differential
migration through a gel matrix.
– Charge/mass ratio
• Requires same charge on all proteins and peptides
– SDS or sodium dodecyl sulfate
• Anionic detergent
• Complexes with the proteins and peptides at a constant ratio of
1:1.4
CE-SDS-Gel Analysis
Molecular Sieving
Detector
capillary filled with entangled
polymer solution
replaceable sieving matrix
improved reproducibility
Separation Mechanism
protein forms complex with SDS
complexes have same charge to size
ratio
separation solely based on the
hydrodynamic size
proteins migrate in order of increasing
size
Detector
ProteomeLabTM SDS-Gel Analysis
Chemistry for the PA800 Plus
SDS-MW Analysis
IgG Purity & Heterogeneity Assay
ProteomeLabTM SDS-MW Analysis
Resolving Power
ProteomeLabTM IgG Purity & Heterogeneity
Resolving Power
ProteomeLabTM SDS-MW Analysis
Kit Components
•
Bare Fused Silica Capillary
– 50 µm ID X 57 cm
•
SDS-MW Gel buffer
• proprietary formulation
• Store at Room Temperature
•
SDS Sample buffer
• 100 mM TrisHCl pH 9.0, 1% SDS
• Store at Room Temperature
•
SDS Protein Sizing Standards
• 10-225 kDa (16 mg/ml)
•
•
10 kDa Internal Size Standard (5 mg/ml)
Acid Wash
• 0.1N HCl
•
Basic Wash
• 0.1N NaOH
ProteomeLabTM SDS-MW Kit
Instrument Setup
• Detection
– PDA @ 220 nm
– 200 m aperture
• Bare Fused Silica Capillary
–
–
–
–
50 m ID
30.2 cm total length (20 cm length to detector)
Capillary ends should be clean, not jagged.
Capillary cut should be perpendicular to capillary length, not
angled
• Interface area should be cleaned before run and after run.
• Interface area must be cleaned after 24 hours of Operation
ProteomeLabTM SDS-MW Kit
Size Standard Preparation
• Remove from refrigerator and allow sit for 15 minutes at room
temperature
• Mix Well by inverting and then centrifuge briefly
• Pipette 10 l of Size Standard into micro vial
• Add 85 l Sample Buffer
• Add 2 l 10 kDa Internal Standard
• Add 5 l 2-mercaptoethanol
• Cap vial and mix thoroughly
• Heat 3 min at 100 C
• Cool in Room Temperature water bath for 5 minutes.
• Pipette 100 l into 200 l pcr vial
ProteomeLabTM SDS-MW Kit
Protein Sample Preparation
• Final protein concentration 0.2 to 2 mg/ml
• Final salt concentration < 50 mM
• If higher reduced loading efficiency
• Desalt sample or use pressure injection
• Reduced Sample
• Dilute sample with at least 50 µl Sample Buffer to a total
volume of 95 µl
• Add 2 µl 10kD Internal Standard
• Add 5 µl 2- mercaptoethanol
• Cap tightly and mix thoroughly
• Heat 3 minutes at 100°C
• Cool in Room Temperature water bath for 5 minutes.
• Pipette 100 l into pcr vial
ProteomeLabTM SDS-MW Kit
Protein Sample Preparation
• NonReduced Sample
• Alkylate minimizes heterogeneity generated by heating
• Dilute sample with at least 50 µl Sample Buffer to a total
volume of 95 µl (0.2 to 2 mg/ml)
• Add 2 µl 10kD Internal Standard
• Add 5 µl 250 mM Iodoacetimide
• Cap tightly and mix thoroughly
• Heat 3 minutes at 70°C
• Cool in Room Temperature water bath for 5 minutes.
• Pipette 100 l into pcr vial
ProteomeLabTM SDS-MW Kit
• Good Resolving Power
• Assay Precision better than 1% for Mobility and
Area%
• Linearity Range 0.2 – 2mg/mL of Total Protein
ProteomeLabTM SDS-MW Kit
Resolving Power
ProteomeLab SDS-MW Kit
Linearity For Sizing Determination
ProteomeLab SDS-MW and IgG Purity
Impurity Determination
ProteomeLabTM IgG Kit
• Separation Mechanism
– Capillary Gel Electrophoresis
– Identical to SDS-MW Separation
• Gel Matrix
– Identical to SDS-MW
• Method optimized for separation of reduced and non
reduced IgG.
• Includes system suitability standard with a specified
amount of non glycosylated heavy chain.
ProteomeLabTM IgG Kit
Kit Components
•
Bare Fused Silica Capillary
– 50 µm ID X 57 cm
•
SDS-MW Gel buffer
• proprietary formulation
• Store at Room Temperature
•
SDS Sample buffer
• 100 mM TrisHCl pH 9.0, 1% SDS
• Store at Room Temperature
•
•
•
IgG Control Standard
10 kDa Internal Size Standard (5 mg/ml)
Acid Wash
• 0.1N HCl
•
Basic Wash
• 0.1N NaOH
ProteomeLabTM IgG Kit
Instrument Setup
• Detection
– PDA @ 220 nm
– 200 m aperture
• Bare Fused Silica Capillary
–
–
–
–
50 m ID
30.2 cm total length (20 cm length to detector)
Capillary ends should be clean, not jagged.
Capillary cut should be perpendicular to capillary length, not
angled
• Interface area should be cleaned before run and after run.
• Interface area must be cleaned after 15 hours of operation
ProteomeLabTM IgG Kit
IgG Control Preparation
• Upon receiving aliquot 95 µl into 0.5 ml microtubes
– Store at -20
•
•
•
•
•
•
•
•
Thaw 1 95 µl aliquot
Add 2 µl 10 kDa Internal Standard
Add 5 µl 2-mercaptoethanol
Cap vial and mix thoroughly
Centrifuge at 300 g for 1 minutes
Heat 10 min at 70 C
Cool in Room Temperature water bath for 5 minutes.
Pipette 100 µl into pcr vial
ProteomeLabTM IgG Kit
IgG Sample Preparation
• Final salt concentration < 50 mM
– If higher reduced loading efficiency
– Desalt sample or use pressure injection
• Reduced Sample
–
–
–
–
–
–
–
–
100 µg IgG to 50 – 95 µl Sample Buffer to a total volume of 95 µl
Add 2 µl 10kDa Internal Standard
Add 5 µl 2-mercaptoethanol
Cap tightly and mix thoroughly
Centrifuge 300g for 1 minute
Heat 10 minutes at 70°C
Cool in Room Temperature water bath for 5 minutes.
Pipette 100 µl into pcr vial
ProteomeLabTM IgG Kit
Protein Sample Preparation
• NonReduced Sample
• Alkylate minimizes fragmentation
• 100 µg IgG to 50 – 95 µl Sample Buffer to a total
volume of 95 µl
• Add 2 µl 10kDa Internal Standard
• Add 5 µl 250 mM Iodoacetimide
• Cap tightly and mix thoroughly
• Centrifuge 300g for 1 minute
• Heat 10 minutes at 70°C
• Cool in Room Temperature water bath for 5 minutes.
• Pipette 100 µl into pcr vial
ProteomeLabTM IgG Kit
Methods
• Run Preconditioning Method
• Run Sequence of 24 injections
– First run is IgG Control Standard as a system
suitability
– 24 is maximum number of injections due to
polymer build up on interface block
– Buffer vials incremented every 8 runs
• Necessary due to polymer build up on the vial cap
ProteomeLabTM IgG Kit
What to expect?
Goodness of fit:
0.999332
ProteomeLabTM IgG Kit
Resolving Power
PDA - 220nm
IgG reduce
PDA - 220nm
IgG reduce
0.06
0.06
0.05
0.05
0.04
0.04
0.03
0.03
0.02
0.02
0.01
0.01
0.00
0.00
8
10
12
14
16
18
20
Minutes
22
24
Human IgG - Reduced: Reproducibility
26
28
AU
AU
PDA - 220nm
IgG reduce
What is the difference between
SDS-MW kit and IgG Purity kit?
• METHODS
– SDS-MW
• Rinses and Gel fill once every 6 samples
– IgG
• Rinses and Gel fill prior to every injection
• Size Standards for SDS MW Kit
• IgG Control Standards for IgG Purity Kit
Tips and Tricks for SDS Gel Analysis
• Recommended protein concentration is 1
mg/ml
– Higher concentrations can result in tailing peaks
due to insufficient protein binding
• Salt concentration should be <50 mM in final
dilution
– Insufficient sample loading
• Gel must be degassed and at room
temperature
– Spikes present on the baseline
Tips and Tricks for SDS Gel Analysis
• Buffer Vial Caps cannot be reused
–
–
–
–
–
Dried polymer is very difficult to remove
Caps degrade over time and with washing
Result is poor injection reproducibility
Broken capillaries
Pressure failures
Tips and Tricks for SDS Gel Analysis
• Interface block must be cleaned before and
after every run
– Polymer precipitates on interface block
– Do not fill buffer vials with more than 1.5 ml of gel
which will minimize polymer on interface block
– Lack of cleaning causes broken capillaries and
pressure failures