Esophageal Adenocarcinoma Methods and Protocols

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2 Materials

1. U9 Buffer: 9 M urea, 2% CHAPS, 3[(3-cholamidopropyl)
   dimethylammonio)]-propanesulfonic acid.


2. Prepare equilibration buffers specific for each protein chip sur-
   face as described in Table 1 (see Note 1). Prepare each buffer
   with and without 0.02% Triton X100 except for H50, where
   no Triton is used (see Note 2).


3. Phosphate buffered saline: 8 g/L NaCl, 0.2 g/L KCl, 1.15 g/L
   Na 2 HPO 4 , 0.2 g/L KH 2 PO4.


4. Sinapic acid matrix: freshly prepared for each assay by adding
   125 μL of 1% trifluoracetic acid and 125 μL of acetonitrile to a
   vial of sinapic acid (supplier, Bio-Rad).


5. All-in-one protein standard 2 (supplier, Bio-Rad).


6. Protein chips (supplier, Bio-Rad):

2.1 Sample
Equilibration
on Protein Chip
Surface

Table 1

Preparation of buffers used for optimization of protein chip binding

conditions

Chip type Equilibration buffer

Q10 and CM10 100 mM sodium acetate, pH 4.0

100 mM sodium acetate, pH 5.0

100 mM sodium phosphate, pH 6.0

100 mM sodium phosphate, pH 7.0

100 mM sodium phosphate, pH 8.0

100 mM Tris–HCl, pH 9.0

IMAC (A)50 mM sodium acetate, 0.2 M sodium chloride,

pH 4.5

(B) Phosphate buffered saline

100 mM copper sulfate

100 mM nickel sulfate

H50 (A) Phosphate buffered saline,100 mM sodium

chloride, 10% acenonitrile

(B) Phosphate buffered saline,100 mM sodium

chloride, 10% acenonitrile, 0.1% trifluoracetate

(C) Phosphate buffered saline,100 mM sodium

chloride, 20% acenonitrile

(D) Phosphate buffered saline,100 mM sodium

chloride, 20% acenonitrile, 0.1% trifluoracetate

Proteomic Protocol