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●● IMAC—immobilized metal affinity capture. These can be
activated using copper or nickel.
●● CM10—weak cation exchanger. This surface binds proteins
that are positively charged at a given pH and is highly pH
dependent.
●● Q10—strong anion exchanger. This surface binds proteins
that are negatively charged at a given pH. It is therefore
also highly pH dependent.
●● H50—hydrophobic interaction. This surface binds proteins
with hydrophobic surface regions.- SELDI-TOF MS Instrument (supplier, Bio-Rad).
- Proteoprep immunodepletion spin column kit (Sigma-
Aldrich). Includes elution buffer, equilibration buffer, and
microcentrifuge tubes. - Off-gel fractionator, immobilized pH gradient (IPG) strips
and materials (Agilent). - Off-gel separation buffer: 25.2 g urea, 9.1 g thiourea, 600 mg
DTT, 6 mL glycerol, 600 μL ampholytes pH 3.0–10.0, in
50 mL deionized water. - Desalt buffer: (10 mM HEPES).
- Vivaspin 3000 molecular weight cut-off (MWCO) spin col-
umn filters. - Sample loading buffer (SDS, Invitrogen).
- 10 mM dithiothreitol.
- 50 mM iodoacetamide.
- nu-PAGE 10% Bis-Tris gels with MES running buffer.
- SeeBlue Plus 2 pre-stained standards.
- Fixing buffer: 50% methanol, 10% acetic acid.
- Coomassie G-250 colloidal stain.
- Harris Unicore 1 mm cutter.
- Stain remover: 50 mM ammonium bicarbonate, 30%
acetonitrile. - Elution buffer: 50% formic acid, 30% acetonitrile, 10%
isopropanol.
3 Methods
The technique of obtaining proteomic profiles of samples by
surface- enhanced laser desorption ionization time-of-flight mass
spectroscopy can be used for the analysis of serum or plasma sam-2.2 Protein
Identification
Peter Kelly