436 T.Marshall Electrophoresis 1983,4,436-438

Thomas Marshall Artefacts associated with Nonidet P-40 upon sodium National Board of Occupational dodecyl sulphate polyacrylamide gel electrophoresis of

protein Safety and Health, Chemistry Division, Solna

One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of protein in the presence of Nonidet P-40 (NP-40) induces vertical streaking which breaks horizontal bands of protein to produce a “multiple spot” ef- fect. The same effect can be induced upon high resolution two-dimensional electro- phoresis if the NP-40 concentration of the first-dimension isoelectric focusing (IEF) gels is increased to 4 % w/v and the gels electrophoresed (SDS-PAGE) without SDS- equilibration. The relevance of this phenomenon as regards interpretation of two- dimensional electrophoretic patterns is discussed.

High resolution two-dimensional electrophoresis [ 1 I com- bines IEF (first dimension) in the presence of 9 M urea and 2 % NP-40 with SDS-PAGE (second dimension) to resolve com- plex protein mixtures [2l. For calibration of molecular weight under these conditions I have been investigating SDS-PAGE of commercial low molecular weight (LMW) calibration pro- teins incorporated into polyacrylamide gel cylinders of IEF gel composition i. e. containing 9 Murea and 2 % NP-40. This has revealed interesting electrophoretic artefacts associated with the presenceofNP-40, which may be significant to thein- terpretation of high resolution two-dimensional electrophore- sis patterns.

The proteins of LMW Electrophoresis Calibration Kits (Phar- macia, Uppsala, Sweden) were prepared for electrophoresis by heating, at 95 “C for 5 min, with 100 p1 of 0.0625 M Tris- HCl, pH 6.8, containing 2 % SDS and 5 % 2-mercapto- ethanol. On cooling, 30 pl of the protein solution were added to 10 ml of IEF gel mixture [ 1,31, prepared by dissolving 5.5 g of urea in a solution comprising 2.0 ml water, 2.0 ml 10 % NP-40, 1.66 ml acrylamide stock (28.38 % acrylamide, 1.62 % bisacrylamide) and 0.5 ml of Ampholine (pH ranges: 2.5-4.0, 3.5-10, 9-11, in a 1:9:1 v/v/v ratio). The mixture was degassed and polymerisation initiated by adding 15 pl of 10 % ammonium persulphate solution and 15 pl of N,N,N‘,N‘-tetramethylethylenediamine (TEMED). The gels were cast in polypropylene drinking straws of approximately 3 mm internal diameter [3 I. Duplicate gels were prepared without the NP-40 which was replaced with 2.0 ml of water. Gel pieces (3 mm length) were electrophoresed in 0.025 M Tris-base, 0.192 Mglycine, 0.1 % SDS with and without prior equilibration (5-60 min) in a solution of 2.3 % SDS, 10 % glycerol in 0.0625 M Tris-HC1, pH 6.8 141. SDS-PAGE was performed, without a stacking gel, on polyacrylamide gradi- ent gels (75 x 75 x 3 mm) prepared with a 4- 10 % polyacryl- amide gradient overthe top 10 mm and alinear 10-20 %poly- acrylamide gradient over the remaining 65 mm. High resolu- tion two-dimensional electrophoresis of human serum was performed as previously described [3,4l except that the first- dimension IEF gels contained either 0,0.5,2.0 or 4.0 % w/v NP-40 and were electrophoresed in the second dimension (SDS-PAGE) without SDS-equilibration. The polypeptides

Correspondence: Dr. T. Marshall, Ph. D., National Board of Occupational Safety & Health, Chemistry Division, S-17184 Solna, Sweden,

Abbreviations: IEF: Isoelectric focusing; NP-40: Nonidet P-40; SDS: Sodium dodecyl sulphate; PAGE: Polyacrylamide gel electrophoresis; LMW: JAW molecular weight

were subsequently detected by silver staining as previously de- scribed 1561.

Fig. 1 shows the electrophoretic patterns obtained for LMW calibration proteins following SDS-PAGE from gel pieces devoid of NP-40 (a) or containing 2 % NP-40 (final con- centration), with (c-g) or without (b) prior equilibration in the SDS solution. NP-40 induces extensive streaking which dis- torts the polypeptide bands to produce a “multiple spot” phenomenon unless the gels are first equilibrated for a minimum period of 1 h (Fig. 1). These artefacts are dependent upon both the concentration of NP-40 and the amount ofpro- tein electrophoresed. Reducing the NP-40 concentration to 0.5 % eliminates both effects. Maintaining the NP-40 con- centration at 2 % whilst reducing the total protein load gradually reduces the intensity of vertical streaking but increases the sharpness of separation of “spots” in each band. The two-dimensional patterns of human serum are shown in Fig. 2. High NP-40 concentration (4 % w/v) induced vertical streaking (Fig. 2A). Streaking was less evident with 2 % w/v

Figure 1 . SDS-PAGE of LMW calibration proteins following incorpora- tion into polyacrylamide gel cylinders containing 9 Mureaeither without (a) or with (b-g) inclusion of 2 % NP-40. Gels(a) and(b) wereelectrophoresed without prior equilibration. The remainder were equilibrated in an SDS- containing solution for either 5 (c), 10 (d), 15 (e), 30 (f) or 60 min (g) prior to electrophoresis. The proteins were: 1, phosphorylase b, MW 94 OOO; 2, bovine serum albumin, 67 OOO; 3, ovalbumin, 43 OOO; 4, carbonic anhy- drase, 30 OOO; 5, soybean trypsin inhibitor, 20 OOO and 6, a-lactalbumin, 14 400. Each tract contained approximately 600 ng of total protein.

0 Verlag Chemie GmbH, D-6940 Weinheirn, 1983 01 73-0835/83/0612-0436S02.50/0

Electrophoresis 1983,4,436-438 Artefacts associated with Nonidet P-40 437

Figure 2. Two-dimensional electrophoreticpatternsofO.1 plhuman serum.TheIEF gelscontainedeither4 %,(A);2 %,(B);0.5 %,(C)orO %,(D)NP-40 w/v and were electrophoresed (SDS-PAGE) without SDS-equilibration. In each case the anode ofthe IEF gel was positioned to the left and electrophoresis performed from top to bottom. Arrowheads indicate polypeptides which appear as “multiple spots” due to vertical streaking induced by NP-40. The posi- tion ofalbumincorresponds tothe prominent spottowardsthecentreofeachgel. TheAmpholine(2 %w/v)wasamixtureofthepH ranges3.5-10and5-7 (9:l VIV).

NP-40 (Fig. 2B) and was virtually eliminated when the NP-40 concentration was reduced to 0.5 % (Fig. 2C) or com- pletely omitted (Fig. 2D). The streaking associated with 4 % NP-40 appeared to induce a “multiple spot” effect in polypeptides (arrowheads, Fig. 2A) and minor indications of

the phenomenon were detected with 2 % NP-40 (arrowheads, Fig. 2B). These effects were eliminated when the IEF gels were SDS-equilibrated for 10-60 min prior to electrophoresis (not shown). High NP-40 concentration also resulted in extensive trailing from albumin which obscured surrounding poly-

43 8 T. Marshall Electrophoresis 1983,4,436-438

peptides (Fig. 2A, B). This was largely eliminated and the compactness and resolution of surrounding polypeptides in- creased by either omitting the NP-40(Fig. 2D) or by reducing its concentration to 0.5 % (Fig. 2C).

A characteristic feature of high resolution two-dimensional electrophoresis is its ability to resolve polypeptides of single charge difference and thereby reveal charge microheter- ogeneity as a string of closely associated spots of similar elec- trophoretic mobility [7,81. A disturbing feature of the vertical streaking induced by NP-40 upon one-dimensional electro- phoresis is that it breaks horizontal bands of protein to produce an apparent “multiple spot” effect (Fig. 1). Although evidence of vertical streaking can be induced upon high resolu- tion two-dimensional electrophoresis (particularly by increas- ing the NP-40 concentration to 4 % and omitting the SDS- equilibration step, Fig. 2A) it does not appear to be a problem, providing the IEF gels are SDS-equilibrated prior to second- dimension SDS-PAGE. Nevertheless, the apparatus and con- ditions employed in our two-dimensional system are rather unique (for example, no stacking gel, relatively thick-3 mm- electrophoresis gels and prolonged SDS equilibration) and therefore, the possibility may still exist that streaking and associated artefacts could arise in other two-dimensional systems, of which there are many [21. Analysis of serum pro- teins in different concentrations of NP-40 should eliminate any uncertainty. It is evident that reducing or eliminating the NP-40 may prove advantageous since the patterns so ob- tained for serum without SDS-equilibration were as good as those obtained with the recommended procedure. Equilibra- tion is inconvenient, time-consuming and prone to protein

losses [ 1 I. Nevertheless, for the moment, this alternative should be approached with caution since serious problems are likely to be encountered upon analysis of detergent-solubilised polypeptides. The interaction of protein with detergent de- pends upon many factors [91. Although the chemical nature of the NP-40 phenomenon has not been investigated, it is my opinion that it may arise through electrophoresis of mixed micelles of SDS and NP-40, accompanied by progressive dis- placement of protein-bound NP-40 by SDS.

I wish to thank Ms. K. M. Williams for heh with preparations of the manuscript and Professor 0. Vesterberg (in receipt of a grant from the Swedish Work Environment Fund) for providing laboratory facilities.

Received July 7, 1983

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