ProteinChip Energy Absorbing Molecules (EAM) ® Instruction Manual Catalog #C30-00001, #C30-00002, #C30-00003, #C30-00004 For technical support, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723).
Introduction ProteinChip energy absorbing molecules (EAM) are an essential component of successful ProteinChip SELDI system experiments. EAM is a generic name for molecules that assist in desorption and ionization of the analyte. Known as “matrix” in traditional MALDI mass spectrometry, EAM are applied in organic solvent, solubilizing many proteins on the array surface. As the EAM dry, the proteins cocrystallize with the EAM.
ProteinChip SPA EAM give better resolution and fewer multiply charged ions than ProteinChip EAM-1 EAM. For protein profiling using only one kind of EAM, ProteinChip SPA EAM are the molecules of choice. ProteinChip EAM-1 EAM, 5 mg/vial ProteinChip EAM-1 EAM are proprietary molecules (MW 231.21) and work well for proteins in the 10–50 kD mass range. ProteinChip EAM-1 EAM prove useful in allowing desorption and ionization of proteins that are difficult to detect, such as glycosylated proteins.
Table 1. Preparation of ProteinChip EAM in solution using commonly used solvent systems. EAM Solvent System ProteinChip CHCA 50% acetonitrile 49.75% HPLC grade H2O Preparation Preparation 1. Add 200 µl solvent to vial (there should be undissolved CHCA in the bottom of the tube) 0.25% trifluoroacetic 2. Mix acid (TFA) 3. Centrifuge 4. Draw off supernatant and dilute with equal part solvent ProteinChip SPA 50% acetonitrile 1. Add 400 µl solvent to vial 49.50% HPLC grade H2O 2. Mix 3. Centrifuge 0.
Solvent systems and ProteinChip EAM in solution are best prepared fresh every day. Acetonitrile volatilizes rapidly, and EAM in solution break down significantly within 24 hours at room temperature. Freezing EAM after dissolving them in solvent may preserve them for about a week; however, it is recommended to prepare fresh solvent and ProteinChip EAM in solution each day. Preparing Saturated Solutions 1. Bio-Rad supplies ProteinChip EAM as 5 mg of dried powder in a tube.
Applying EAM to Arrays The method of ProteinChip EAM application to the array surface requires practice as it can significantly influence the quality of the data. A minute volume (0.3–1.0 µl) of ProteinChip EAM in organic solvent must be applied to each sample prior to analysis. It is recommended that ProteinChip EAM be applied to the spots after they have dried completely for the greatest spot-to-spot consistency.
Ordering Information Catalog # Description C30-00001 ProteinChip CHCA Energy Absorbing Molecules (EAM), 5 mg/vial, 20 ProteinChip SPA Energy Absorbing Molecules (EAM), 5 mg/vial, 20 ProteinChip EAM-1 Energy Absorbing Molecules (EAM), 5 mg/vial, 20 ProteinChip Energy Absorbing Molecules (EAM) Kit, includes 6 vials each CHCA, EAM-1, and SPA EAM C30-00002 C30-00003 C30-00004 MicroMix is a trademark of Diagnostic Products Corporation.
Bio-Rad Laboratories, Inc. Web site www.bio-rad.
