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Keck
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2D-LC
Two dimensional chromatofocusing/non-porous
reverse phase HPLC protein separation (2D-LC)
Two dimensional chromatofocusing/non-porous
reverse phase HPLC protein separation (2D-LC). In this approach the
chromatofocusing first dimension separates proteins based on
isoelectric point (pI), and subsequent fractions are sequentially and
automatically separated by surface hydrophobicity on a reverse-phase
column. Two dimensional chromatofocusing and nonporous reverse phase
HPLC will be carried out on the Keck Lab’s Beckman-Coulter Proteomelab
PF2D System which utilizes a self-contained liquid flow path, thereby
reducing the risk of sample contamination during transfer between
dimensions. The chromatofocusing is performed on a 2.1 x 250 mm HPCF-1D
column (Eprogen, Inc.) using a pH monitor and UV detector at 280 nm.
Buffers for chromatofocusing are (A) 25 mM Bis-Tris, 6 M urea, and 0.2%
n-octyl β-D-glucopyranoside (pH 8.5) and (B) polybuffer 74
(diluted 1:10 with water), 6 M Urea, and 0.2% n-octyl β-D-glucopyranoside
(pH 4.0). The HPCF-1D column is first equilibrated with buffer A until
the pH of the effluent is 8.5. Approximately 0.2 to 5 mg of protein
sample (as determined by amino acid analysis) is injected on the system
which is then washed with buffer A for 20 minutes. Next, a 115 minute
0-100% pH gradient is run at 200 µl/min with fractions collected at
0.3-pH intervals. The nonporous reverse-phase HPLC separation is
performed at 50°C on a 4.6 x 33 mm HPCF2D column (Eprogen, Inc.)
equilibrated at a flow rate of 1 ml/min for 8 minutes with 0.1% TFA
(Buffer A). The first dimension fraction is injected and after a 2 min
wash with buffer A, the proteins are eluted using a 30 minute (or
longer for complex samples) 0-100% Solvent B (100% Acetonitrile, 0.08%
TFA) linear gradient. Proteins are detected at 214 nm and collected for
tryptic digestion and MS/MS analysis.
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