Calculating Isoelectric Point

[MedPR]

When doing electrophoresis, given the pH and either the direction of the anode/cathode or the direction that an amino acid migrates, you can tell if the amino acid is acidic, basic, or nonpolar.

However, can you calculate the isoelectric point?

For instance, in electrophoresis with media pH of 6.0, glutamic acid will migrate towards the anode, since the carboxyl groups will deprotonate and thus the glutamate will have a net negative charge. Based on that, given an image of the electrophoresis results, you can guess that if only one of a set of unknown amino acids (of which one unknown is glutamic acid) migrates towards the anode, that it is glutamic acid.

Is there any way to calculate the pI? If not, what would need to be given to calculate the pI?

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[aSagacious]

I haven't done one of these in a while, but if I remember correctly you need a 'ruler' to estimate pI. In other words in one lane you have a series of known amino acids (with known isoelectric points) and in the other lane your unknown sample. You then compare the migration distance of the unknown to your 'ruler' to gauge an approximate pI.

Edit: You can also titrate with a strong base and look for the vertical portions (poor buffering regions) of the curve to approximate pI.

 

[MedPR]

Well I know that you can find it based on a graph, but I was mainly asking given only the information in the OP.

 

[aSagacious]

In that case you'd stick to the first part of my answer, the isoelectric focusing gel:

 

[MedPR]

I'm confused by your image as well (sorry!).

What is the pH of that gel? I guess it might make my question more clear if you see what made me ask it.

http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/proteins.htm

If you scroll about a third of the way down the page, there is an animation of electrophoresis.

Is it possible to have determined the pI's given?

 

[Class1P]

Biologist here:

If you're doing 2d electrophoresis of proteins the gel and IP are separate.

Usually you place your proteins on a strip with a varying acidity. Run a current through it and your proteins separate based on isoelectric point. Then you take the strip out, lay it sideways on top of an acrylamide gel. You then run a current through that and your proteins separate by size.. What you end up with is a gel with proteins in it separate by size on one axis and by isoelectric point on the other axis.