For help: how to determine the pI in the IPG strip?
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Wed Dec 28 04:59:49 EST 2005
doctorjiangsheng at gmail.com wrote:
> I know that polyacrylamide gel
> electrophoresis (PAGE) can determine pI easily.
Electrophoretic separations can be designed to separate by isoelectric
point (isoelectric focussing), by molecular mass (SDS-PAGE), or by
Stokes radius (DISK-electrophoresis).
> But we have no
> equipment. I only can determine pI by means of IPG strip.
Then you have very good equipment, otherwise you'd have to cast tube
gels for IEF yourself.
> I find the voltage can not over 1000.00 volt. Do you know what the
> electric voltage and the hours are needed?
Pharmacia reports 10,000 Vh, although I found that with large membrane
proteins 25,000 Vh gives better results. With a 1,000 V source, try an
overnight run, if that doesn't suffice, try 24 h.
> So I think it is impossible to calculate pI
> according the protein sequence.
That is correct, because the pI of amino acids was measured in water,
not in a protein where interaction with other charged groups can
significantly alter the pI (by several pH units).
> Does every one ever determine the pI of beta-lactamase using IPG strip?
As others have suggested, you can use pI-marker protein (available from
Pharmacia, BioRad, Sigma and others) and focus them. Then make a
calibration curve of position vs pI of the marker. This should result in
a straight line. Given the position of your lactamase on the strip you
can then easily calculate the pI.
Alternatively, cut the strip into small pieces (say, 5 mm long) and
incubate each of them with a little water. Then measure the pH of that
water and plot pH vs piece number. Again you should get a straight line,
from which you can read the pH of the piece where your protein was.
A more old-fashioned way is to use horizontal electrophoresis on
cellulose acetate or even filter paper. You need several strips soaked
in buffers of different pH. The protein is added to a marked spot in the
middle between cathode and anode. After electrophoresis the migration
distance is measured (counting movement towards the cathode negative,
towards the anode positive). Plot migration distance vs pH and
interpolate the pH at which no movement would occur. This is your pI.
This type of experiment can be performed with home-made equipment even
in very poor institutions.
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