Hans-Peter Kost Serva-Feinbiochcbmica,Heidelberg
Evaluation of isoelectric focusing runs as specific resistance/volthowr plots Isoelectric focusing (IEF) runs, e.g. on ultrathin gels, are characterized by an extensive change of gel electric parameters, caused by the rearrangement of carrier ampholyte components from a uniform distribution to a highly ordered pH gradient. A particularly important parameter is the specific resistance Q [Ohm*cm] which has been determined in polymerization mixtures (with and without carrier ampholytes) and in 125 X 0.15 m m ultrathin gels, pH 3-10 with 5%T, 3 % c , 5% Servalyt carrier ampholytes, pH 3-10. T h e starting specific resistance @ of ultrathin IEF gels, calculated from the geometric gel dimensions and electric current values (V, mA), is in agreement with the data determined directly in 30 mL samples of polymerization mixtures by using a conductivity meter. Electric specific conductivity/Volthour (Vh) plots proved to be a valuable tool for the evaluation of gel systems with and without protein samples during IEP runs. These plots are usually S-shaped, indicating that the key part of pH gradient formation takes places in a relatively short time. A “good” ultrahin gel, after a short lag phase, shows a rapid increase in specific resistance due to a rapid pH gradient formation and a slope of about 18 Ohm*cm/Vh. IEF is finished in about 3000 Vh. After prolonged gel storage, and especially in partially dried gels, the electrical parameters approach equilibrium only slowly, as indicated by the relatively shallow slope (8.9 Ohm*cm/Vh). Accordingly, separations need more than 4000 Vh. In gels with salt containing samples, after normal IEF for about 1750 Vh, cracks appear, often followed by sparking at about 2700 Vh, accompanied by a sharp (often intermittent) drop in specific resistance.
In the past. performance characteristics of electrophoretic materials have mostly been judged by the final results obtained. The finished pherogram (fixed, stained, destained and dried) especially of isoelectric focusing (IEF) gels (e.g. [l-31) is still the ultimate control of a run. Alterations and improvements have mostly been made on an experimental basis and to a lesser extent due to theoretical considerations [4].The evaluation of key electric parameters already during a run is a valuable complimentary tool for gel and sample evaluation. IEF runs are characterized by an extensive change of gel electrical parameters, caused by the rearrangement of carrier ampholyte components from a uniform distribution to a highly ordered pH gradient [4]. Also, electrode processes and electrode polarization, etc., play a role. Most of today’s power supplies are equipped with volthour (Vh) integrators; constant maximum values for voltage (volts), current (milliamperes) and power (watts) m a y be preset. To register all values, a constant reading is required. The knowledge of @, the specific electrical resistance of the specimen as a material parameter, and the constant preset data, I (max.), U (max.) [or N (max.)], are sufficient to calculate all parameters which are nonliiniting at the time of data readout. These make it possible to judge gel polymerization mixtures, and, as a function of the Vh product, to evaluate electrophoretic runs, especially IEF.
scribed previously [3]. Ultrathin gels were cast as described [I], with slight modifications, with thicknesses of 150 pm. Polymerization mixtures of 30 mL for conductivity measurements were thermostatized using a Haake T P 42, Typ F 4291 thermostatized water bath set at 25” C. Conductivities were determined using a Hanna Instruments H I 8633 Conductivity Meter which was repeatedly calibrated with 0.1 Mol/L KCI (12.88 niS/cm at 25”C).The ultrathin gels used for most experiments were 125 X 125 X 150 mm, with 5%T. 3%C, 5 % Servalyt carrier ampholytes, pH 3-10. Specific resistances in the gel were determined using the equation: Q
=
U*q/(l*l)
where U is the voltage (V), I the current [mi%], I the separation distance in [cm] and q the cross-section area i n [cm2].
___ Correspondence: Dr. Hans-Peter Kost, Serva Feinbiochemica. Carl-BenzStraBe 7, DW-6000 H e i d e l b e r g , Germany
Degassed mixtures ofacrylamide monomer kept at 25“ C remain relatively stable at low values of specific electric conductivity. When N,N,N’,N’-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) are added, the conductivity increases sharply to the polymerization starting value (ca. 900 W c m ) and after a short lag phase of about 2-3 min, further increases over a period of about 90 min. The conductivity stabilizes after about 100 min (ca. 1400 pS/cm)(Fig. 1). At the same time, the gel obtains its final consistency. The increase in conductivity may be explained by the formation of sulfate ions from peroxodisulfate, when the polymerization “catalyst” APS initiates the reaction. Five percent carrier ampholyte in the gel mixture not only,increase the starting conductivity to around 1500 pS/cm but also “buffer” conductivity changes to about only 15% (results not shown).
Abbreviations: C, cruss-linker concentration (O/O); IEF, isoelectric focusing; @, specific resistant-, [kOhm*cm]; T, total acrylarnide concentration ( O h ) ; Vh, volthour product
When carrying out runs, the starting specific resistance is in agreement with the data determined di-
All chemicals were analytical grade or of the highest available purity from Serva Feinbiochemica, if not otherwise mentioned. The electrophoresis equipment was as de-
6 VCH ~erl;igsgesellscliallmbH, D-6940 Weinheim, 1992
0 l73-0835/92/09 10-0660 $3 .jO+.28 10
Evaluation of runs by resistancefvolthour plots
Electruphorrsis 1992, 13, 660-661
661
rectly from the polymerization mixture using a conductivity meter. However, one has to keep in mind the influence of the measuring temperature (25”C versus 4“ C) and also that a conductivity meter is operated by AC (alternating current), in order to avoid electrode polarization effects, whereas IEF gels are operated by DC (direct current). Polarization effects increase the specific resistance. Figure 2, I
Spec Conductivity 6 0 0
1
8
/
(VS I c m )
7
_-
I
/
800
1000 600
2000 Temperature: 4 0 C
400
1
200
1
0_ i0
4000 JUdt [Vhl
Figure 3. Salt-containing samples (protein test mixture 9 Serva, 20 mg/ mL in distilled water with 2% NaC1, 10 pL per sample slot) were applied (at around 150 Vh). After normal IEF for about 1750 Vh, cracks begin to form in the gel layer, which eventually lead to spark formation at 2700Vh. After a normal increase the specific resistance drops sharply.
, 20
3000
40
60 Time (minutes)
80
100
120
&.
Temp 25OC
Figure I. Conduclivity [bS/cm] of a polymerization mixture ( 5 %T, 3O/oC, no carrier ampholyte) as a function of time. After a short lag phase, the conductivity rises monotonously and stabilizes after about 100 min at 1400 [pS/cm]. The gel at this time has reached its final consistency.
(K 0hm.cm) 2o ~r
1 ’good’l
15
, / ’
l/r______’
/’ 10
curve 1 depicts the data of a “good” gel with rapid pH gradient generation and a slope of about 18 [Ohm*cm/volthour] in the linear part of the S-shaped curve. IEF is finished in about 3000Vh. When standard protein mixtures or other proteins are run on such a gel, a perfect separation results (not shown, for previous publications see [1-3]). In Fig. 2, curve 2, separations required more than 4000 Vh. In such a case the cause often lies in partial gel drying prior to separation (e.g., gel exposed for more than about 10-20 min). Some commercial outdated ready-to-use gels may exhibit similar behavior. In curve 3, salt-containing samples have been applied (see text; at around 150 Vh). After normal IEF for about 1750 Vh, cracks appear in the gel layer, eventually resulting in sparking at 2700 Vh. After a normal rise, the specific resistance drops sharply (see text). The quality of an IEF run, depending on both gel and sample, may be judged using Vh/specific resistance plots. This additional evaluation criterion, besides staining gels, is a valuable tool for experienced and also inexperienced gel users, as well as for quality control of gels and samples.
5
I
1000
3000
2000 TBmPer.lYle:
4-
c
Received August 10, 1992
4000 I U d t [Vhl
Figure 2. Comparison of the Vh versus specific resistance [kOhm*cm] plots of 125 X 125 X 0.15 m m gels (SVoT, 3 % c , 5% Servalyt pH 3-10). A “good” gel (upper graph) exhibits a rapid pH gradient formation and a slope of about 18 Ohm*cm/Vh in the linear part of the curve, indicating that IEF may be finished at around 3000 Vh. Lower graph: Old ( e.g. storage for 2 years] as well as partially dried gels (e.g., gel exposed for more than about 10-20 min) show a much more shallow slope and reach equilibrium much later.
References [ l ] Radola, B.J., Elrcirophoresis 1980, I , 43-56. [2] Kinzkofer, A. and Radola, B.J., Electrophoresis 1981, 2, 174-183. [3] Kost, H.-P. and Scheibe, S . , in B.J. Radola (Ed.): Electrophoresis Forum ’89, Technical University Munich 1989, pp. 335-339. [4] Righetti, P.G., Isoelectric Focusing: T h e o q Methodology and Applications. Elsevier Biomedical Press. Amsterdam 1987.
Evaluation of isoelectric focusing runs as specific resistance/volthowr plots Isoelectric focusing (IEF) runs, e.g. on ultrathin gels, are characterized by an extensive change of gel electric parameters, caused by the rearrangement of carrier ampholyte components from a uniform distribution to a highly ordered pH gradient. A particularly important parameter is the specific resistance Q [Ohm*cm] which has been determined in polymerization mixtures (with and without carrier ampholytes) and in 125 X 0.15 m m ultrathin gels, pH 3-10 with 5%T, 3 % c , 5% Servalyt carrier ampholytes, pH 3-10. T h e starting specific resistance @ of ultrathin IEF gels, calculated from the geometric gel dimensions and electric current values (V, mA), is in agreement with the data determined directly in 30 mL samples of polymerization mixtures by using a conductivity meter. Electric specific conductivity/Volthour (Vh) plots proved to be a valuable tool for the evaluation of gel systems with and without protein samples during IEP runs. These plots are usually S-shaped, indicating that the key part of pH gradient formation takes places in a relatively short time. A “good” ultrahin gel, after a short lag phase, shows a rapid increase in specific resistance due to a rapid pH gradient formation and a slope of about 18 Ohm*cm/Vh. IEF is finished in about 3000 Vh. After prolonged gel storage, and especially in partially dried gels, the electrical parameters approach equilibrium only slowly, as indicated by the relatively shallow slope (8.9 Ohm*cm/Vh). Accordingly, separations need more than 4000 Vh. In gels with salt containing samples, after normal IEF for about 1750 Vh, cracks appear, often followed by sparking at about 2700 Vh, accompanied by a sharp (often intermittent) drop in specific resistance.
In the past. performance characteristics of electrophoretic materials have mostly been judged by the final results obtained. The finished pherogram (fixed, stained, destained and dried) especially of isoelectric focusing (IEF) gels (e.g. [l-31) is still the ultimate control of a run. Alterations and improvements have mostly been made on an experimental basis and to a lesser extent due to theoretical considerations [4].The evaluation of key electric parameters already during a run is a valuable complimentary tool for gel and sample evaluation. IEF runs are characterized by an extensive change of gel electrical parameters, caused by the rearrangement of carrier ampholyte components from a uniform distribution to a highly ordered pH gradient [4]. Also, electrode processes and electrode polarization, etc., play a role. Most of today’s power supplies are equipped with volthour (Vh) integrators; constant maximum values for voltage (volts), current (milliamperes) and power (watts) m a y be preset. To register all values, a constant reading is required. The knowledge of @, the specific electrical resistance of the specimen as a material parameter, and the constant preset data, I (max.), U (max.) [or N (max.)], are sufficient to calculate all parameters which are nonliiniting at the time of data readout. These make it possible to judge gel polymerization mixtures, and, as a function of the Vh product, to evaluate electrophoretic runs, especially IEF.
scribed previously [3]. Ultrathin gels were cast as described [I], with slight modifications, with thicknesses of 150 pm. Polymerization mixtures of 30 mL for conductivity measurements were thermostatized using a Haake T P 42, Typ F 4291 thermostatized water bath set at 25” C. Conductivities were determined using a Hanna Instruments H I 8633 Conductivity Meter which was repeatedly calibrated with 0.1 Mol/L KCI (12.88 niS/cm at 25”C).The ultrathin gels used for most experiments were 125 X 125 X 150 mm, with 5%T. 3%C, 5 % Servalyt carrier ampholytes, pH 3-10. Specific resistances in the gel were determined using the equation: Q
=
U*q/(l*l)
where U is the voltage (V), I the current [mi%], I the separation distance in [cm] and q the cross-section area i n [cm2].
___ Correspondence: Dr. Hans-Peter Kost, Serva Feinbiochemica. Carl-BenzStraBe 7, DW-6000 H e i d e l b e r g , Germany
Degassed mixtures ofacrylamide monomer kept at 25“ C remain relatively stable at low values of specific electric conductivity. When N,N,N’,N’-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) are added, the conductivity increases sharply to the polymerization starting value (ca. 900 W c m ) and after a short lag phase of about 2-3 min, further increases over a period of about 90 min. The conductivity stabilizes after about 100 min (ca. 1400 pS/cm)(Fig. 1). At the same time, the gel obtains its final consistency. The increase in conductivity may be explained by the formation of sulfate ions from peroxodisulfate, when the polymerization “catalyst” APS initiates the reaction. Five percent carrier ampholyte in the gel mixture not only,increase the starting conductivity to around 1500 pS/cm but also “buffer” conductivity changes to about only 15% (results not shown).
Abbreviations: C, cruss-linker concentration (O/O); IEF, isoelectric focusing; @, specific resistant-, [kOhm*cm]; T, total acrylarnide concentration ( O h ) ; Vh, volthour product
When carrying out runs, the starting specific resistance is in agreement with the data determined di-
All chemicals were analytical grade or of the highest available purity from Serva Feinbiochemica, if not otherwise mentioned. The electrophoresis equipment was as de-
6 VCH ~erl;igsgesellscliallmbH, D-6940 Weinheim, 1992
0 l73-0835/92/09 10-0660 $3 .jO+.28 10
Evaluation of runs by resistancefvolthour plots
Electruphorrsis 1992, 13, 660-661
661
rectly from the polymerization mixture using a conductivity meter. However, one has to keep in mind the influence of the measuring temperature (25”C versus 4“ C) and also that a conductivity meter is operated by AC (alternating current), in order to avoid electrode polarization effects, whereas IEF gels are operated by DC (direct current). Polarization effects increase the specific resistance. Figure 2, I
Spec Conductivity 6 0 0
1
8
/
(VS I c m )
7
_-
I
/
800
1000 600
2000 Temperature: 4 0 C
400
1
200
1
0_ i0
4000 JUdt [Vhl
Figure 3. Salt-containing samples (protein test mixture 9 Serva, 20 mg/ mL in distilled water with 2% NaC1, 10 pL per sample slot) were applied (at around 150 Vh). After normal IEF for about 1750 Vh, cracks begin to form in the gel layer, which eventually lead to spark formation at 2700Vh. After a normal increase the specific resistance drops sharply.
, 20
3000
40
60 Time (minutes)
80
100
120
&.
Temp 25OC
Figure I. Conduclivity [bS/cm] of a polymerization mixture ( 5 %T, 3O/oC, no carrier ampholyte) as a function of time. After a short lag phase, the conductivity rises monotonously and stabilizes after about 100 min at 1400 [pS/cm]. The gel at this time has reached its final consistency.
(K 0hm.cm) 2o ~r
1 ’good’l
15
, / ’
l/r______’
/’ 10
curve 1 depicts the data of a “good” gel with rapid pH gradient generation and a slope of about 18 [Ohm*cm/volthour] in the linear part of the S-shaped curve. IEF is finished in about 3000Vh. When standard protein mixtures or other proteins are run on such a gel, a perfect separation results (not shown, for previous publications see [1-3]). In Fig. 2, curve 2, separations required more than 4000 Vh. In such a case the cause often lies in partial gel drying prior to separation (e.g., gel exposed for more than about 10-20 min). Some commercial outdated ready-to-use gels may exhibit similar behavior. In curve 3, salt-containing samples have been applied (see text; at around 150 Vh). After normal IEF for about 1750 Vh, cracks appear in the gel layer, eventually resulting in sparking at 2700 Vh. After a normal rise, the specific resistance drops sharply (see text). The quality of an IEF run, depending on both gel and sample, may be judged using Vh/specific resistance plots. This additional evaluation criterion, besides staining gels, is a valuable tool for experienced and also inexperienced gel users, as well as for quality control of gels and samples.
5
I
1000
3000
2000 TBmPer.lYle:
4-
c
Received August 10, 1992
4000 I U d t [Vhl
Figure 2. Comparison of the Vh versus specific resistance [kOhm*cm] plots of 125 X 125 X 0.15 m m gels (SVoT, 3 % c , 5% Servalyt pH 3-10). A “good” gel (upper graph) exhibits a rapid pH gradient formation and a slope of about 18 Ohm*cm/Vh in the linear part of the curve, indicating that IEF may be finished at around 3000 Vh. Lower graph: Old ( e.g. storage for 2 years] as well as partially dried gels (e.g., gel exposed for more than about 10-20 min) show a much more shallow slope and reach equilibrium much later.
References [ l ] Radola, B.J., Elrcirophoresis 1980, I , 43-56. [2] Kinzkofer, A. and Radola, B.J., Electrophoresis 1981, 2, 174-183. [3] Kost, H.-P. and Scheibe, S . , in B.J. Radola (Ed.): Electrophoresis Forum ’89, Technical University Munich 1989, pp. 335-339. [4] Righetti, P.G., Isoelectric Focusing: T h e o q Methodology and Applications. Elsevier Biomedical Press. Amsterdam 1987.
