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

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

Separation Mechanism

capillary filled with entangled

polymer solution

replaceable sieving matrix

improved reproducibility

Detector

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


• 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


• 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




• Pipette 100 l into pcr vial


• Good Resolving Power

• Assay Precision better than 1% for Mobility and Area%

• Linearity Range 0.2 – 2mg/mL of Total Protein


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.


Kit Components


– 50 µm ID X 57 cm

• SDS-MW Gel buffer • proprietary formulation


• SDS Sample buffer • 100 mM TrisHCl pH 9.0, 1% SDS


• IgG Control Standard

• 10 kDa Internal Size Standard (5 mg/ml)

• Acid Wash • 0.1N HCl

• Basic Wash • 0.1N NaOH


Instrument Setup

• Detection

– PDA @ 220 nm

– 200 m aperture


– 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


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


• Pipette 100 µl into pcr vial


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


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


• Centrifuge 300g for 1 minute



• Pipette 100 µl into pcr vial


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

Goodness of fit:

0.999332


What to expect?


Resolving Power

Minutes

8 10 12 14 16 18 20 22 24 26 28

AU

0.00

0.01

0.02

0.03

0.04

0.05

0.06

AU

0.00

0.01

0.02

0.03

0.04

0.05

0.06

PDA - 220nm

IgG reduce

PDA - 220nm

IgG reduce

PDA - 220nm

IgG reduce

Human IgG - Reduced: Reproducibility

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


• 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


• 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

capillary electrophoresis of proteins -...

Documents