Reversible Negative Staining of Protein on Electrophoresis Gels
39
l. Load the sample directly onto the reversed-phase half of the biphasic column
or,
2. assemble and 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.
3. Pump 0.1 % TFA through the cartridge to obtain a stable baseline.
4. Load the protein eluate into the loop and inject it at a flow of 0.1-0.2 mL/min.
After the absorbance value has returned to baseline, pump 0.1 % TFA at 0.2ml/
min for 10 mins.
5. Remove the column from the cartridge and assemble it in the sample loading
device.
6. Dry the column with pressurised nitrogen to store until sequencing.
Comments
i) The minor amount of SDS that co-elutes with the protein does not interfere
with an efficient retention of the protein to PVDF or the Hewlett-Packard
sequencing column, making additional steps for removal of SDS unnecessary.
ii) Retention on Immobilon PSQ membrane at different loading flows was determined with four radioiodinated proteins (BSA, streptokinase, cytochrome c
and insulin, 100 picomole each) eluted from SDS-PA gels. For loading flows
between 25 and 500 Ill/min, more than 90 % of the eluted protein was
retained on the PVDF disk. At ImL/min or higher flows, significant amounts
of proteins passed through the membrane.
iii) The capacity of the membrane disk (6 mm 2 ) under the loading conditions
here described was about 400 picomole.
iv) For the Hewlett-Packard sequencing columns, flow rate has little effect on
protein retention, however care should be taken not to overpressurize the
column and create leaks.
Advantages
This procedure permits the concentration of low ~bundant proteins eluted from
several gel bands by filtering the solution through the sequencing membrane or
applying directly to protein sequencing columns. This is accomplished by using
standard HPLC equipment. It is an alternative to other established procedures
(vacuum loading, centrifugal filtration). The advantages of the present technique
are:
(a) a precise control of the loading flow on the membrane (a factor affecting protein retention on the membrane),
(b) it is an unattended operation (particularly useful when loading large volumes
of very diluted samples),
(c) it allows to record the time-course for loading and membrane clean-up
through UV detection, and
(d) allows for the rapid and efficient desalting of samples with minimal sample
loss.
39
l. Load the sample directly onto the reversed-phase half of the biphasic column
or,
2. assemble and 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.
3. Pump 0.1 % TFA through the cartridge to obtain a stable baseline.
4. Load the protein eluate into the loop and inject it at a flow of 0.1-0.2 mL/min.
After the absorbance value has returned to baseline, pump 0.1 % TFA at 0.2ml/
min for 10 mins.
5. Remove the column from the cartridge and assemble it in the sample loading
device.
6. Dry the column with pressurised nitrogen to store until sequencing.
Comments
i) The minor amount of SDS that co-elutes with the protein does not interfere
with an efficient retention of the protein to PVDF or the Hewlett-Packard
sequencing column, making additional steps for removal of SDS unnecessary.
ii) Retention on Immobilon PSQ membrane at different loading flows was determined with four radioiodinated proteins (BSA, streptokinase, cytochrome c
and insulin, 100 picomole each) eluted from SDS-PA gels. For loading flows
between 25 and 500 Ill/min, more than 90 % of the eluted protein was
retained on the PVDF disk. At ImL/min or higher flows, significant amounts
of proteins passed through the membrane.
iii) The capacity of the membrane disk (6 mm 2 ) under the loading conditions
here described was about 400 picomole.
iv) For the Hewlett-Packard sequencing columns, flow rate has little effect on
protein retention, however care should be taken not to overpressurize the
column and create leaks.
Advantages
This procedure permits the concentration of low ~bundant proteins eluted from
several gel bands by filtering the solution through the sequencing membrane or
applying directly to protein sequencing columns. This is accomplished by using
standard HPLC equipment. It is an alternative to other established procedures
(vacuum loading, centrifugal filtration). The advantages of the present technique
are:
(a) a precise control of the loading flow on the membrane (a factor affecting protein retention on the membrane),
(b) it is an unattended operation (particularly useful when loading large volumes
of very diluted samples),
(c) it allows to record the time-course for loading and membrane clean-up
through UV detection, and
(d) allows for the rapid and efficient desalting of samples with minimal sample
loss.