ProteinChip CM10 Array (Weak Cation Exchanger) ® Instruction Manual Catalog #C57-30075 For technical support, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723).
Uses n Protein profiling and biomarker discovery n Rapid protein analysis to determine purity, mass confirmation, or both How It Works The operating mechanism of ionic exchange ProteinChip arrays is the reversible binding of charged molecules to the surface, and the property of a peptide or protein that governs its binding is its net surface charge. Since surface charge is the result of weak acidic and basic amino acids within the protein, binding of the protein to the array is highly pH dependent.
ProteinChip cassette-compatible bioprocessor (catalog #C50-30011). It is not necessary to remove the arrays when using the cassettecompatible bioprocessor; however, individual arrays can be removed if needed. To do this, remove the bioprocessor reservoir before taking any arrays out of the cassette. Be careful not to touch the spots on the array. A pair of ProteinChip array forceps (catalog #C20-10002) helps effectively remove the arrays from the cassette (see Figure 2). Fig. 1.
Recommended Binding and Washing Buffers n ProteinChip CM low-stringency buffer (catalog #K20-00003) 0.1 M sodium acetate, pH 4.0 n ProteinChip CM high-stringency buffer (catalog #K20-00004) 50 mM HEPES, pH 7.0 n Sodium/ammonium acetate buffer (10–100 mM), pH 4–6 n Sodium/ammonium phosphate buffer (10–100 mM), pH 6–8 n Tris-HCl buffer (10–100 mM), pH 7.5–9.0 Fractionation for Serum Profiling Fractionation of serum prior to profiling is recommended.
4. Remove the binding buffer from the wells, and add 150–250 µl deionized (DI) water to each well; remove immediately. Repeat once. 5. Remove the reservoir from the bioprocessor base clamp assembly. 6. Air-dry the arrays for 15–20 minutes. 7. Add ProteinChip energy absorbing molecules (EAM) after removing the reservoir; use the cassette hold-down frame provided with the cassette-compatible bioprocessor to keep the cassette flat during EAM addition. 8. Apply 1 µl of ProteinChip EAM in solution to each spot.
Protocol 2: Serum Profiling On-Spot 1. Prewet the spots with 5 µl of binding buffer for 5 minutes. Repeat once. 2. Remove the prewetting solution and replace with 5 µl of sample. Do not allow the spot to air-dry during sample application. 3. Incubate in a humid chamber for 30 minutes with shaking (on Micromix shaker setting 20/4). 4. Wash each spot with 5 µl of binding buffer with shaking and remove buffer. Repeat two more times. 5. Wash each spot with 5 µl of DI water. Repeat once. 6.
MicroMix is a trademark of Diagnostic Products Corporation. The SELDI process is covered by US patents 5,719,060, 6,225,047, 6,579,719, 6,818,411, and other issued patents and pending applications in the US and other jurisdictions. © 2006 Bio-Rad Laboratories, Inc.
Bio-Rad Laboratories, Inc. Web site www.bio-rad.
