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For help: how to determine the pI in the IPG strip?

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  #1  
Old 12-20-2005, 03:02 AM
doctorjiangsheng@gmail.com
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Default For help: how to determine the pI in the IPG strip?




Dear Sir or Madam:
Hi! I am studying beta-lactamase now. I want to determine the pI using
IPG strip. I do IEF using pH range 3-6 IPG strip. I have focused the
beta-lactamase. It is on the point which is 7.4cm from the anode point.
The whole strip distance is 17cm. What is the pI of the beta-lactamase?

I am looking forward for your reply.
Sincerely yours,
Sheng Jiang
2005-12-20

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  #2  
Old 12-20-2005, 04:21 AM
DK
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Default For help: how to determine the pI in the IPG strip?

In article <1135047721.652920.242770@z14g2000cwz.googlegroups .com>, "[Only registered users see links. ]" <[Only registered users see links. ]> wrote:

You must include known pI standards in the same run.

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  #3  
Old 12-20-2005, 06:27 AM
doctorjiangsheng@gmail.com
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Default For help: how to determine the pI in the IPG strip?

But as far as I know in immobilized pH gradient strip the pH of every
point is already known. I think the company which manufacture it must
know the pH of every point in the IPG strip.

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  #4  
Old 12-20-2005, 06:49 PM
Wolfgang Schechinger
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Default For help: how to determine the pI in the IPG strip?

Dear Sheng Jiang,

Dima is right. You always need to include appropriate controls. Many
thigs could have happened to your strips that altered the pH. If you
need to soak them in water or a buffer before use, you could add some
pH indicator dye to check that eversything is ok.

To check your experiment, just cut out small pieces of the buffer
strips and measure their pH with pH-paper.

Finally, as long you were analyzing native bLa, you might be lucky and
find the pI in databases like Expasy or Swissprot. There are tools you
can use to calculate the pI from a given protein sequence.

Best,

Wolfgang

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  #5  
Old 12-21-2005, 04:36 AM
doctorjiangsheng@gmail.com
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Default For help: how to determine the pI in the IPG strip?

Dear Sir or Madam:
First of all please allow me to express my gratitude to all of you!
I have come up with a lot of difficulties in my determination of pI of
beta-lactamase. I am in China and there are even no people who ever
determine the pI of protein. I know that polyacrylamide gel
electrophoresis (PAGE) can determine pI easily. But we have no
equipment. I only can determine pI by means of IPG strip.
I have failed several times in determining pI of beta-lactamase using
IPG strip. I prepared my beta-lactamase by means of sonication.
I rehydrate my IPG strip (pH 3-6) in crude sonicated bacterial enzyme
extracts. I use bio-rad IEF protein cell for iso-electric focusing.
I find the voltage can not over 1000.00 volt. Do you know what the
electric voltage and the hours are needed?
I am piloting inhibitor-resistant TEM (IRT) in China. It is one of
beta-lactamases, which has never found in China. I have read many
papers on it. Never has anyone purified beta-lactamases and then
determine the protein sequence. And then according to the protein
sequence calculate the pI. So I think it is impossible to calculate pI
according the protein sequence.
Does every one ever determine the pI of beta-lactamase using IPG strip?
Can he help me?
My telephone: 86-28-85423212,
My Fax: 86-28-85423212
My E-mail: [Only registered users see links. ]
I am looking forward for your reply.
Sincerely yours,
Sheng Jiang
2005-12-21

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  #6  
Old 12-28-2005, 09:59 AM
Dr Engelbert Buxbaum
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Default For help: how to determine the pI in the IPG strip?

[Only registered users see links. ] wrote:


Electrophoretic separations can be designed to separate by isoelectric
point (isoelectric focussing), by molecular mass (SDS-PAGE), or by
Stokes radius (DISK-electrophoresis).


Then you have very good equipment, otherwise you'd have to cast tube
gels for IEF yourself.


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.


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).


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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  #7  
Old 12-28-2005, 05:21 PM
doctorjiangsheng@gmail.com
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Default For help: how to determine the pI in the IPG strip?

Dear Dr Engelbert Buxbaum:
Thank you very much! I have read your message carefully. I will go on
to try to determine pI using IPG strip.
Sincerely yours,
Sheng Jiang
2005-12-29

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