38
L. CASTELLANOS-SERRA et aI.
iv} To collect the solution, centrifuge only for about 30 s-l min.
v} To minimize losses due to adsorption onto surfaces, proteins analysed with
this protocol are only in contact with polypropylene (two 1.S-mL tubes and
the syringe); in our experience when working at the low picomole level, significant losses can arise when using centrifugal concentration. If necessary,
concentrate by vacuum-centrifugation (Savant) or, alternatively, for protein
sequencing of highly diluted samples, apply the solution to PVDF by the
"chromatographic loading" procedure (see 2.7).
vi} For the extraction of highly hydrophobic proteins: prepare the formic acidisopropanol-acetonitrile solution just before use and do not store it in polypropylene tubes, in order to avoid polymer leakage from the tube to the solution that will generate artifacts detectable by mass spectrometry.
Advantages
This is a simple procedure, which does not require dedicated expensive equipment for achieving a rapid elution of proteins with very high yields even at the
low picomole level. The high elution yields obtained in the absence of detergents
in the elution buffer make this procedure particularly useful for protein elution
for further microanalysis. When combined with the chromatographic loading
onto PVDF ( see 2.7), it allows the recovery and concentration of low abundant
proteins from several lanes/spots.
2.7
Chromatographic Concentration on PVDF Disks
or Hewlett-Packard Sequencing Columns
of Low-Abundant Proteins Eluted From Gel Bands
Procedure (Castellanos-Serra et al. 7997)
PVDF-disks (Materials: Sequencing membrane (Problot, or Immobilon PSQ),
zero dead volume on-line filter cartridge (Knauer, Supelco), Teflon O-ring}.
1. Cut a disk of sequencing membrane to fit the internal diameter of the zerodead volume cartridge (for the Knauer cartridge, 6-mm diameter).
2. Wet the membrane with methanol and water. Insert the membrane disk into
the cartridge and retain it in place with the aid of the Teflon O-ring.
3. Connect the cartridge to the HPLC injector provided with a 5 mL loop (fIlled
with 0.1 % aqueous TFA) and to the detector set at 215 nm.
4. Pump 0.1 % TFA through the cartridge to obtain a stable baseline.
5. Load the protein eluate into the loop and inject it at a flow of 0.1-0.2 mLimin.
6. After the absorbance value has returned to baseline, pump 0.1 % TFA for
about 10 mins.
7. Remove the membrane from the cartridge and store at 4°C until sequencing.
Hewlett-Packard sequencing column (Materials: Hewlett-Packard reversed-phase
sequencing column (top half of the biphasic column), Hewlett-Packard sequencer
sample loading funnel, or a Hewlett-Packard sequencing column zero dead volume adapter}.
L. CASTELLANOS-SERRA et aI.
iv} To collect the solution, centrifuge only for about 30 s-l min.
v} To minimize losses due to adsorption onto surfaces, proteins analysed with
this protocol are only in contact with polypropylene (two 1.S-mL tubes and
the syringe); in our experience when working at the low picomole level, significant losses can arise when using centrifugal concentration. If necessary,
concentrate by vacuum-centrifugation (Savant) or, alternatively, for protein
sequencing of highly diluted samples, apply the solution to PVDF by the
"chromatographic loading" procedure (see 2.7).
vi} For the extraction of highly hydrophobic proteins: prepare the formic acidisopropanol-acetonitrile solution just before use and do not store it in polypropylene tubes, in order to avoid polymer leakage from the tube to the solution that will generate artifacts detectable by mass spectrometry.
Advantages
This is a simple procedure, which does not require dedicated expensive equipment for achieving a rapid elution of proteins with very high yields even at the
low picomole level. The high elution yields obtained in the absence of detergents
in the elution buffer make this procedure particularly useful for protein elution
for further microanalysis. When combined with the chromatographic loading
onto PVDF ( see 2.7), it allows the recovery and concentration of low abundant
proteins from several lanes/spots.
2.7
Chromatographic Concentration on PVDF Disks
or Hewlett-Packard Sequencing Columns
of Low-Abundant Proteins Eluted From Gel Bands
Procedure (Castellanos-Serra et al. 7997)
PVDF-disks (Materials: Sequencing membrane (Problot, or Immobilon PSQ),
zero dead volume on-line filter cartridge (Knauer, Supelco), Teflon O-ring}.
1. Cut a disk of sequencing membrane to fit the internal diameter of the zerodead volume cartridge (for the Knauer cartridge, 6-mm diameter).
2. Wet the membrane with methanol and water. Insert the membrane disk into
the cartridge and retain it in place with the aid of the Teflon O-ring.
3. Connect the cartridge to the HPLC injector provided with a 5 mL loop (fIlled
with 0.1 % aqueous TFA) and to the detector set at 215 nm.
4. Pump 0.1 % TFA through the cartridge to obtain a stable baseline.
5. Load the protein eluate into the loop and inject it at a flow of 0.1-0.2 mLimin.
6. After the absorbance value has returned to baseline, pump 0.1 % TFA for
about 10 mins.
7. Remove the membrane from the cartridge and store at 4°C until sequencing.
Hewlett-Packard sequencing column (Materials: Hewlett-Packard reversed-phase
sequencing column (top half of the biphasic column), Hewlett-Packard sequencer
sample loading funnel, or a Hewlett-Packard sequencing column zero dead volume adapter}.