EDC Impressum & DSE

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Dr. Hanspeter Schickle
Wolfgang Gstrein

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Agarose -  Separations

September 21, 2020

Isoelectric focusing is the only method to separate different IgG molecules from each other. In all other methods, such as SDS- and native electrophoresis, the whole IgG spectrum appears as 1 broad band.
The IEF-run
Because of its high molecular weight (160 kD) oligoclonal IgG is not easy to focus in sharp bands. Normally a pH-gradient of 3-10 should be run more than 4000Vh.
Agarose gels cannot survive this procedure. Because of intrinsic fixed negative charges in agarose gels - caused by carboxylic and sulfonic residues - there is an electroendosmotic water transport towards the cathode. This part of the gel becomes more and more wet and the anodal part will become thinner and thinner.
 After about 1500 Vh the IEF with Agarose gel must be stopped. This leads to blurred IgG bands with not enough sharpness. Polyacrylamide-based gels (PAG-gels) can be run up to 4500 Vh. This is enough for displaying even the finest bands.
Please compare identical CSF-sample on Polyacrylamide- and Agarose gels below.

IEF on PAG and Agarose: One identical sample run on both types of gels

IEF: PAG versus Agarose

Separation on Agarose-Gels, 2 competitors:
(Same samples run also on: General Silver stain)