Guide to Isoelectric Focusing

1 APB, RW 5/5/981 Guide to Isoelectric FocusingIntroductionIsoelectric Focusing methods are widely applied for the separation of proteins, peptides andenzymes. The principle: In a pH gradient the sample components migrate towards the anodeor the cathode to the pH values, where their net charges are zero: their Isoelectric points (pI).Should a protein diffuse away from its pI, it would gain a charge and migrate back: this is thefocusing effect. Much more details of the theory and practice of Isoelectric Focusing can befound in the book by Righetti [1] .The proteins are driven to their Isoelectric points by the electric field. The Isoelectric pointsof the proteins can be estimated with a calibration curve using marker proteins. Native ordenaturing conditions can be chosen by omitting or adding high amounts of proteins become highly concentrated at their pIs.

2 This results in a high sensitivity fordetection. Small charge differences can be differentiated. For improvement of resolution,narrow gradients can be employed. The method is also suitable for preparative applications,when high amounts of proteins - one hundred and more micrograms - have to be applications require different Isoelectric Focusing methods. Amersham Biosciences offers a wide range of products for Isoelectric Focusing . In the following technicalnote the various methods and products for Isoelectric Focusing are Focusing (IEF) is mostly run in horizontal gels because of several reasons:- As IEF separates only according to the charge, the gel matrix must contain large pore a soft gel can slide down between vertical glass plates. The gel should preferably be caston a film Isoelectric Focusing requires efficient cooling and exact temperature control (see below).

3 This is optimally achieved on a horizontal ceramics cooling plate connected to a thermostaticcirculator, or a peltier cooling Samples have to be applied on a defined pH location within the pH gradient in order to avoidBIEF in immobilized pH gradient of cheese proteins,courtesy: I. Krause FML-W eihenstephan, FRGAPB, RW 5/5/982aggregation and precipitation of some proteins. This is only possible on a horizontal gel withan open Because sharply focused bands can only be obtained with a high field strength, high voltageshave to be applied. Only horizontal equipment can meet the necessary safety power supply must provide enough voltage for obtaining sharply focused IEF zonesand must preferably be programmable in order to run a multiphase IEF protocol normal sized IEF (ca. 10 cm and longer separation distances) a modular system isavailable.

4 IEF in mini gels (4 cm separation distance) can be run in an automatedelectrophoresis unit containing the separation chamber, programmable power supply andstaining II IEF unitcode no. 18-1018-06 EPS 3500 XL Power Supplycode no. 19-3500-01 MultiTemp III Thermostatic Circulatorcode no. 18-1102-78(230 V) MultiTemp III Thermostatic Circulatorcode no. 18-1102-77(115 V) PhastSystemall in onecode no. 18-1018-24(230 V)PhastSystemall in onecode no. 18-1018-23(115 V) Isoelectric Focusing with Ampholine and Pharmalyte Ampholine and Pharmalyte are mixtures of 600 to 700 different homologues ofamphoteric compounds with a spectrum of Isoelectric points between 3 and 10 form a pHgradient under the influence of the electric field. These substances have high bufferingcapacities at their Isoelectric points.

5 They have molecular weights below 1 kDalton and do notbind to proteins, because they are highly hydrophilic. Their general name is carrierampholytes . Mixtures with narrow intervals are available for higher resolution and theselection of defined Isoelectric point difference between Ampholine and Pharmalyte is based on the chemistry of theirproduction. Ampholine are produced by reacting aliphatic oligoamines with acrylic acids,whereas Pharmalyte are co-polymerisates of glycine, glycylglycine, amines andepichlorhydrin. Thus the Isoelectric points and buffering properties of the individualhomologues of the different products are slightly bottles contain a 40 % w/v stock solution of carrier ampholytes; a 2 to 3 % w/vconcentration is required in the gels for Isoelectric Focusing .

6 Pharmalyte bottles contain ansimilar amount of carrier ampholytes like Ampholine bottles, it is not exactly defined. Thebuffering capacities of the individual homologues within a mixture are differing to the instructions they are used in a dilution of 1 in 16 volumes, which is equal toAmpholine .Ampholine preblended can be used directly for agarose , Ultrodex or polyacrylamide gelsand form linear no. 80-1127-1525 mLpH no. 80-1127-1725 mLpH no. 80-1127-1925 mLAmpholine broad range are used as the backbone for custom designed pH gradients, inorder to obtain optimised resolution for certain no. 80-1125-8725 mLAPB, RW 5/5/983 Ampholine narrow range are narrow intervals, which are used to blend custom designedpH no. 80-1125-8925 mLpH no. 80-1125-9025 mLpH no.

7 80-1125-9125 mLpH no. 80-1125-9225 mLpH no. 80-1125-9325 mLpH no. 80-1125-9425 mLMixing table for Ampholine , volumes for 30 mL monomer solution, examples:pH rangepH mL mLpH mL mLpH mL mLPharmalyte broad ranges can be used directly for agarose , Ultrodex or polyacrylamidegels and form linear 5code no. 17-0451-0125 mLpH 4 no. 17-0452-0125 mLpH 5 8code no. 17-0453-0125 mLpH 8 no. 17-0455-0125 mLpH 3 10code no. 17-0456-0125 mLMixing table for Pharmalyte , volumes for 30 mL monomer solution, examples:pH range:pH 5pH 4 5 8pH 9pH 8 3 10 mL mL mLpH 5 10 mL mLpH 8 mL mLpH 4 9 mL mLpH 5 9 mL mLpH mL mLpH mL mLPharmalyte narrow ranges are intervals for special dedicated applications inpolyacrylamide gels, indicated in the following list:pH no.

8 17-0562-0125 mL ( 1-antitrypsin)pH no. 17-0563-0125 mL (transferrin)pH 5 6code no. 17-0564-0125 mL ( )pH no. 17-0566-0125 mL (hemoglobin)Ampholine and Pharmalyte can also be blended with each other in order to obtainmixtures with more different homologues, if , RW 5/5/984 Electrode solutionsTo maintain a stable gradient, filter paper strips soaked in electrode solutions are appliedbetween the gel and the electrodes, an acid solution is used at the anode and a basic one at thecathode. Should, for example, an acidiv carrier ampholyte reach the anode, its basic moietywould become a positively charged and it would migrate back towards the cathode. Theelectrode solutions are particularly important for long separations in gels containing urea, forbasic gradients and short pH intervals.

9 They are not necessary for short gels, like PhastGels .Suggestions of different solutions are given in the following chapters for different gel electrode stripscode no. 18-1004-40100 stripsPlateau phenomenonIn general, problems with carrier ampholytes can arise when long Focusing times arenecessary. For example, in the case of short intervals or in the presence of highly viscousadditives such as urea or non-ionic detergents, the gradient slowly starts to drift in bothdirections but particularly towards the cathode. This leads to a plateau in the middle of thegradient with gaps in the conductivity. Part of the proteins leave the gel. It is thus important tolimit the Focusing time and to run similar experiments for exactly the same time forreproducible applicationThe samples are applied on the surface of the gels.

10 Either they are pipetted on piecesfrom cellulose-cotton, which are placed on the gel surface at the optimal pH location, or asilicon applicator strip with sample holes is laid on the gel surface. The latter are availablefor 26 (up to 40 L) or 52 (up to 20 L) samples, the holes are arranged in the distances, orhalf the distances respectively, of the 96 well microtiter plates for multiple syringes . Themode of sample application is dependent on the sample composition and the IEF gel the gradient is sensitive to high salt and buffer concentrations, it is very helpful todesalt the samples before application. Desalting columns for sample preparation: PD-10columns prepacked with Sephadex columns code no. 17-0851-0130 IEF sample application pieces (20 L)code no. 80-1129-46200 IEF/SDS sample appl.