Protein Protocols Protein Protocols

276 Patton
device (CCD) camera. The tremendous improvement in detection sensitivity and linear
dynamic range certainly justifies the investment in equipment. Procedures for the detection
of electrophoretically separated proteins utilizing colorimetric and luminescent
metal chelate stains are presented in this chapter. Methods for the elution of the metal
chelate stains are also presented. The procedures are applicable for detection of proteins
or peptides in polyacrylamide or agarose gels as well as on nitrocellulose, PVDF, or
nylon membranes. Owing to the electrostatic mechanism of the protein visualization
methods, metal chelate stains are unsuitable for detection of proteins and peptides
immobilized on cationic membranes.
2. Materials
2.1. Colorimetric Detection of Electroblotted Proteins on Membranes
1. Block buffer: 0.1% polyvinylpyrrolidone-40 (PVP-40) in 2% glacial acetic acid.
2. Ferrozine–ferrous stain (stable for at least 6 mo at room temperature): 0.75 mM 3-(2pyridyl)-5,6-bis
(4-phenylsulfonic acid)-1,2,4-triazine disodium salt (Ferrozine), 30 mM
ferric chloride, 15 mM thioglycolic acid in 2% glacial acetic acid. Alternatively, commercially
prepared stain solutions of Ferrozine–ferrous (Rev–Pro stain kit; Genomic Solutions,
Ann Arbor, MI) or Pyrogallol Red-molybdenum (Microprotein-PR kit; Sigma
Chemical Company, St. Louis, MO) can be used.
3. 2% Glacial acetic acid.
4. Ferrocyanide–ferric stain (stable for at least 6 mo at room temperature): 100 mM sodium
acetate, pH 4.0; 100 mM potassium ferrocyanide, 60 mM ferric chloride.
2.2. Luminescent Detection of Electroblotted Proteins
Using Bathophenanthroline Disulfonate-europium (SYPRO Rose Stain)
1. Formate buffer: 100 mM formic acid, pH 3.7, 100 mM sodium chloride.
2. Bathophenanthroline–europium blot stain (stable for at least 6 mo at room temperature):
1.5 mM bathophenanthroline disulfonic acid disodium salt, 0.5 mM europium chloride,
and 0.2 mM EDTA (added from 1000× stock, pH 7.0).
2.3. Luminescent Detection of Electroblotted Proteins
Using SYPRO Rose Plus Protein Blot Stain
1. SYPRO Rose Plus protein blot stain kit (Molecular Probes, Inc. cat. #S-12011) The kit
contains the following components:
SYPRO Rose Plus blot wash solution (component A), 200 mL
SYPRO Rose Plus blot stain solution (component B), 200 mL
SYPRO Rose Plus blot destain solution (component C), 200 mL
The kit contains sufficient material for staining 10–40 minigel electroblots or four largeformat
electroblots (20 × 20 cm). The SYPRO Rose Plus solutions may be reused up to
four times with little loss in sensitivity.
2.4. Luminescent Detection of Electroblotted Proteins
Using SYPRO Ruby Protein Blot Stain
1. SYPRO Ruby protein blot stain (Molecular Probes, Inc.) is provided in a unit size of
200 mL. The 200-mL volume is sufficient for staining 10–40 minigel electroblots or
four large-format electroblots (20 × 20 cm). SYPRO Ruby protein blot stain may be
reused up to four times with little loss in sensitivity.
2. 7% Acetic Acid, 10% methanol.