Capillary Electrophoresis

For use with the IgG Purity/Heterogeneity and SDS MW Kits.

10 kDa internal standard is used as a mobility marker and all protein mobility is calculated relative to this internal standard.

Accurate determination of a protein’s charge heterogeneity helps establish identity and stability. Capillary Isoelectric Focusing (cIEF) is a powerful technique that allows quantitative analysis of a protein’s isoelectric point (pI). In cIEF, a mixture of sample and ampholyte is introduced into a capillary and subjected to electrophoretic separation. In this process, a pH gradient through which analytes migrate to their respective pI is formed. 

The PA 800 Plus Pharmaceutical Analysis System automates the advanced cIEF technology necessary to successfully achieve high precision and quantitative separations. The use of optimized universal methods and synthetic pI markers attains the highest levels of precision in pI calculation and direct isoform quantitation with on-line UV detection.

This kit provides reagent volumes for analysis of 100 samples and includes: 
• eCap Neutral Capillary, 1 unit 
• cIEF gel 
• cIEF Peptide Marker Kit, 5 vials (pI 4.1, pI 5.5, pI 7.0, pI 9.5, pI 10.0), 240 µL each 
• Sample Loading Solution (SLS), 6 mL 
• CE Grade Water  
 
NOTE: Additional reagents from other vendors are required for this application. Methods and necessary supplies can be referenced in the following AIBs, available for download at www.sciex.com:
• A-11634: Identification of System Parameters Critical for High-Performance cIEF
• A-12015: A Robust cIEF Method: Intermediate Precision for the pH 5-7 Range
• A-12026: High-Resolution cIEF of Therapeutic Monoclonal Antibodies: A Platform Method Covering pH 4-10

Cooling liquid refill for use on temperature controlled instruments, for all Sciex CE systems including the BioPhase, PA 800 Plus, CESI 8000 Plus and P/ACE MDQ instruments (Qty: 1).

Kit Component of P/N 390953 and P/N A10663 (IgG Purity/Heterogeneity Kit.)
For use as an experimental control when separating reduced or non-reduced IgG, with a designated quantity of non-glycosylated heavy chain to test both the resolution and quantitation suitability of the assay prior to running unknowns.

Includes IgG Control Standard 1-pack and all chemistries.
The IgG Purity/Heterogeneity Assay has been developed for researchers employed in industrial biotechnology who are developing and manufacturing IgG reagents for research, diagnostic and therapeutic use. 

This assay has been specified for use on the PA 800 Plus to assess the purity and heterogeneity of IgG reagents in both a reduced and non-reduced state. The methodology involves heat-denaturing a specified concentration of IgG (both reduced and non-reduced) in the presence of SDS, and separating these proteins by size using high-resolution capillary gel electrophoresis technology. This assay will detect impurities as low as 0.1% and includes an IgG control with a designated quantity of non-glycosylated heavy chain to test both the resolution and quantitation suitability of the assay prior to running unknowns
 
Assay chemistry includes:
• Separation Capillary, 57 cm x 50 µm ID bare fused-silica, (2)
• SDS Gel Separation Buffer (proprietary formulation), 140 mL
• SDS Sample Buffer, 100 mM Tris-HCl, pH 9.0/1% SDS, 50 mL
• IgG Control Standard, 1 mg/mL in SDS sample buffer , 1 mL
• Internal Standard, 10 kDa protein, 5 mg/mL, 0.4 mL
• Acidic Wash Solution, 0.1 N HCl, 100 mL
• Basic Wash Solution, 0.1 N NaOH, 100 mL
• IgG Purity and Heterogeneity Analysis Guide, (1)

LIF Test Mix, Sodium Fluorescein (20 mL). Test mix for testing the Laser-Induced Fluorescence (LIF) detector and determining the calibration correction factor (CCF) for the system.

Molecular Weight standards (10 to 225 KDa), 16mg/mL (100 µL) for use with the SDS-MW Kit (Part Number 390953) to provide a sizing ladder to reference proteins of interest against.

No more need to mount capillaries. Pre-assembled EZ-CE cartridge (part # A55625) for multiple applications (Purity Analysis, Rapid Charge Variant Analysis, and Fast Glycan Analysis)
> Precision cut tips – ensure uniformity of injection plug from capillary to capillary
> Alignment precision at window – reduces potential failure due to misalignment
> Reduce preparation time for operator – simplifying the process and training requirements

eCAP™ Neutral Coated Capillary with laser burned window - 50 µm ID x 67 cm total length.
Capillary used with the Advanced cIEF Starter Kit (Part number A80976).
Capillaries must be trimmed to optimize for speed/resolution.
This capillary is also suitable for other applications where no EOF is desired. Thanks to the polyacrylamide coating, no EOF is present. The capillary can be used with both UV and LIF detection systems but capillary life is impacted when used with diodearray detection systems.

The SDS-Gel MW Analysis Kit is designed for the separation and sizing of protein–SDS complexes using a replaceable gel matrix. The gel is formulated to provide an effective protein sieving range of approximately 10 to 225 kDa. Within this size range, the logarithm of protein molecular mass is linear with its reciprocal electrophoretic mobility, allowing the molecular weight of an unknown protein to be estimated from a standard curve of known protein sizes. This chemistry can also be used to effectively quantify the amount of protein and to determine the purity of a protein product.

This includes:
• Separation Capillary, 57 cm x 50 µm ID, bare fused-silica, (2)
• SDS Gel Separation Buffer, 140 mL
• SDS Sample Buffer, 100 mM Tris-HCl, pH 9.0/1% SDS, 50 mL
• SDS Protein Sizing Standard (10 to 225 kDa), 16 mg/mL, 100 µL
• Internal Standard, 10 kDa protein, 5 mg/mL, 0.4 mL
• Acidic Wash Solution, 0.1 N HCl, 100 mL
• Basic Wash Solution, 0.1 N NaOH, 100 mL
• SDS-Gel MW Analysis Guide (1)

SDS-MW Gel Buffer is a 4-pack of the SDS-MW gel used in the SDS-MW Kit (Part Number 390953) and the IgG Purity and Heterogeneity Kit Assay (Part Number A10663).

SDS-MW gel designed for the separation and sizing of protein samples, utilizing SDS complexes as a replaceable gel matrix.