ProteinChip IMAC30 Array (Immobilized Metal Affinity Capture) ® Instruction Manual Catalog #C57-30078 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 Analysis of phosphorylated peptides and proteins n Analysis of histidine (His)-tagged proteins n Analysis of metal-binding proteins n On-chip optimization of purification process to be transferred to IMAC sorbent How It Works The operating mechanism of the ProteinChip IMAC30 array is the reversible binding of proteins to the surface through a coordinated metal interaction.
Technical Considerations n Use ProteinChip IMAC30 arrays with Cu2+ metal for general protein profiling, Ni2+ metal for capture of His-tagged recombinant proteins, and Ga3+ or Fe3+ for phosphorylated peptide and protein capture Figure 1. ProteinChip cassette and reservoir. Figure 2. Removal of ProteinChip arrays from cassette using array forceps. n A sodium acetate wash is necessary when charging arrays with copper.
Fractionation for Serum Profiling Fractionation of serum prior to profiling is recommended; the fractionation procedure produces 6 fractions containing proteins separated on the basis of their isoelectric point. Fractionation allows segregation of highly abundant proteins into a limited number of fractions, thereby reducing signal suppression effects on lower-abundance proteins.
7. Remove the samples from the wells, and wash each well with 150–250 µl binding buffer for 5 minutes, with agitation. Repeat two more times. 8. Remove the binding buffer from the wells and add 150–250 µl DI water to each well; remove immediately. Repeat once. 9. Remove the reservoir from the bioprocessor base clamp assembly. 10. Air-dry the arrays for 15–20 minutes. 11. Add ProteinChip sinapinic acid (SPA) energy absorbing molecues (EAM) (catalog #C30-00002) after removing the reservoir.
8. Wash each spot with 5 µl of binding buffer with shaking, and remove buffer. Repeat two more times. 9. Wash each spot with 5 µl of DI water. Repeat once. 10. Air-dry the array for 15–20 minutes. 11. Apply 1 µl of ProteinChip SPA EAM in solution to each spot. Air-dry for 5 minutes and apply another 1 µl of EAM in solution. Allow to air-dry. 12. Analyze the array using the ProteinChip SELDI system.
BioSepra is a trademark of Pall Corporation. 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. This product involves a license from Hoffmann-La Roche, Inc., Nutley, NJ and/or Hoffmann-LaRoche Ltd., Basel, Switzerland and is provided only for use in research.
Bio-Rad Laboratories, Inc. Web site www.bio-rad.
