Capillary Electrophoresis of Proteins

Size: px

Start display at page:

Download "Capillary Electrophoresis of Proteins"

Tiffany Horton
6 years ago
Views:

1 Capillary Electrophoresis of Proteins SDS Capillary Gel Electrophoresis SDS-CGE

2 Outline CE-SDS Gel Analysis Description of Technique Method Development Tips PA800 plus kits SDS-MW IgG Purity & Heterogeneity

3 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

4 CE-SDS-Gel Analysis Molecular Sieving Detector capillary filled with entangled polymer solution replaceable sieving matrix improved reproducibility Separation Mechanism Detector 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

5 ProteomeLab TM SDS-Gel Analysis Chemistry for the PA800 Plus SDS-MW Analysis IgG Purity & Heterogeneity Assay

6 ProteomeLab TM SDS-MW Analysis Resolving Power

7 ProteomeLab TM IgG Purity & Heterogeneity Resolving Power

8 ProteomeLab TM 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 kda (16 mg/ml) 10 kda Internal Size Standard (5 mg/ml) Acid Wash 0.1N HCl Basic Wash 0.1N NaOH

9 ProteomeLab TM SDS-MW Kit Instrument Setup Detection 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

10 ProteomeLab TM 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

11 ProteomeLab TM 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

12 ProteomeLab TM 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

13 ProteomeLab TM SDS-MW Kit Good Resolving Power Assay Precision better than 1% for Mobility and Area% Linearity Range 0.2 2mg/mL of Total Protein

14 ProteomeLab TM SDS-MW Kit Resolving Power

15 ProteomeLab SDS-MW Kit Linearity For Sizing Determination

16 ProteomeLab SDS-MW and IgG Purity Impurity Determination

ProteomeLab TM IgG Kit Separation Mechanism Capillary Gel Electrophoresis Identical to SDS-MW Separation Gel Matrix Identical to SDS-MW Method

17 ProteomeLab TM 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.

18 ProteomeLab TM 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

19 ProteomeLab TM IgG Kit Instrument Setup Detection 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

20 ProteomeLab TM 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

ProteomeLab TM 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

21 ProteomeLab TM 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 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

22 ProteomeLab TM IgG Kit Protein Sample Preparation NonReduced Sample Alkylate minimizes fragmentation 100 µg IgG to 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

23 ProteomeLab TM 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

24 ProteomeLab TM IgG Kit What to expect? Goodness of fit:

25 ProteomeLab TM IgG Kit Resolving Power

26 AU AU PDA - 220nm IgG reduce PDA - 220nm IgG reduce PDA - 220nm IgG reduce Minutes Human IgG - Reduced: Reproducibility

27 What is the difference between SDS-MW kit and IgG Purity kit? METHODS SDS-MW IgG Rinses and Gel fill once every 6 samples Rinses and Gel fill prior to every injection Size Standards for SDS MW Kit IgG Control Standards for IgG Purity Kit

28 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

29 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

30 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

IgG Purity/Heterogeneity and SDS-MW Assays with High- Speed Separation Method and High Throughput Tray Setup

IgG Purity/Heterogeneity and SDS-MW Assays with High- Speed Separation Method and High Throughput Tray Setup High Throughput Methods to Maximize the Use of the PA 800 Plus system Jose-Luis Gallegos-Perez