Application Note: CSE Separation of Cancer Cell Lysate No. 9.200411 Rev. A 2004 Target Discovery, Inc. Target Discovery, Inc. 4015 Fabian Way Palo Alto, CA 94303 Tel: 650-812-8100 www.targetdiscovery.com/eotrol
Application Note: No. 9.200411 Application Challenge: Separation by Size Analyte: Barrett's Esophageal Epithelial Cell Homogenate Product Family: UltraTrol Dynamic Precoatings Product Used: UltraTrol LN CSE SEPARATION OF CANCER CELL LYSATE James R. Kraly, Norman J. Dovichi Department of Chemistry, University of Washington, Seattle, WA, USA Sizing proteins (i.e., the measurement of protein molecular weight) has been achieved by a variety of analytical methods. The most common method is gel electrophoresis, which is still widely used within the life science community despite its relative high cost in time and resources. During the last two decades, a more efficient and effective alternative: capillary sieving electrophoresis (CSE), 1 also known as capillary gel electrophoresis (CGE), has been demonstrated. This method applied the same principles (and in some instances, the same type of separation matrix) as traditional gel electrophoresis. However, CSE provides a number of advantages over gel-based methods, such as low sample and reagent requirements, rapid separation, inline detection, and ease of automation. In fact, these same methods enabled and expedited the human genome and other large-scale DNA sequencing projects. Size separation of other biomolecules is also of crucial importance for the manufacturing and quality control of biologics for the pharmaceutical industry. Electroosmotic flow is one of the key factors that need to be regulated during a CSE separation. Electroosmotic flow regulation is particularly important when using lower viscosity sieving matrices. We compared and contrasted the effectiveness of bare silica capillaries with an UltraTrol LN dynamic precoating and capillaries with a static polyacrylamide coating, as described by Hjertén, 2 for separating proteins by CSE. Both types of coatings provided similar levels of electroosmotic flow suppression. Nearly identical CSE profiles of proteins from a cancer cell homogenate were obtained from both the dynamically precoated capillary and the static polyacrylamide-coated capillary (Figure 1). We also found that the dynamically pre-coated capillary provided a calibration curve (Figure 3) that was highly linear and comparable to that of the statically coated capillary (Figure 2). The clear advantage of the UltraTrol LN dynamic coating is that both the coating, and any adsorbed proteins that would interfere with run-to-run reproducibility, can be stripped between runs by simple rinse procedures. The dynamic coating can then be replenished by simple rinsing procedures. The polyacrylamide-coated capillary cannot withstand a stripping rinse between runs because it can not be replenished. 1 Rev. A
Figure 1. CSE separation of esophageal cancer cell homogenate using either a polyacrylamide coated capillary (red) or a fused silica capillary precoated with UltraTrol LN (blue). Separation Buffer: 5% dextran (513 kda), 100 mm Ches-Tris, 3.5 mm SDS. Separation Conditions: Electrokinetic injection (1 kv, 3 s). Separation at 25 kv, reverse polarity. Capillary: 20 cm x 30 µm coated, 22 cm x 30 µm bare silica. Figure 2. CSE of eight molecular weight standards using a polyacrylamide coated capillary. Inset shows the linearity of migration time vs. log MW. Separation Buffer: 5% dextran (513 kda), 100 mm Ches-Tris, 3.5 mm SDS. Separation Conditions: Electrokinetic injection (1 kv, 3 s). Separation at 20 kv, reverse polarity. Capillary: 34 cm x 30 µm. Figure 3. CSE of eight molecular weight standards using a bare silica capillary precoated with UltraTrol LN. Inset shows the linearity of migration time vs. log MW. Separation Buffer: 5% dextran (513 kda), 100 mm Ches-Tris, 3.5 mm SDS. Separation Conditions: Electrokinetic injection (1 kv, 3 s). Separation at 20 kv, reverse polarity. Capillary: 25 cm x 30 µm 2 Rev. A
EXPERIMENTAL Separation Methodology All CE analyses were carried out in a custom-made capillary electrophoresis unit. 3,4 A voltage program was used to separate peaks. Capillaries were preconditioned according to the manufacturer s recommendations. The capillaries were rinsed with water for 5 min and separation buffer for 5 min between runs in experiments where polyacrylamide coatings were used. The capillaries were rinsed with 0.5 M NaOH for 5 min and water for 2 min between runs in experiments where UltraTrol LN was used. The capillaries were then precoated with 1X UltraTrol LN in water, and then rinsed with separation buffer for 2 minutes prior to beginning an analysis. All rinses were done at 10 psi. Sample Preparation Barrett's Esophageal cellular homogenates were first denatured in 1% SDS at 95 C for 4 min. Homogenate proteins were then labeled with 100 nmole of the fluorogenic reagent 3-(2- furoyl)quinoline-2-carboxaldhyde (FQ) in 2.5 mm KCN at 65 C for 5 min. Labeled samples were then diluted to 200 L with the CSE separation buffer (100 mm Ches, 100 mm Tris, 3.5 mm SDS) REFERENCES [1] Hjertén S. High-performance electrophoresis: the electrophoresis counterpart of high performance liquid chromatography. J Chromatogr, 1983, 270:1. [2] Hjertén S. High performance electrophoresis. Elimination of electroendosmosis and solute adsorption. J Chromatogr, 1985, 347:191-198. [3] Michels DA, Hu S, Shoenherr RM, Eggertson MJ, Dovichi NJ. Fully automated twodimensional capillary electrophoresis for high sensitivity protein analysis. Mol Cell Proteomics, 2002, 1:69-74. [4] Michels DA, Dambrowitz KA, Eggertson MJ, Lauterbach K, Dovichi NJ. Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate. Electrophoresis, 2004, 25:3098-3015. 3 Rev. A