152
Electrophoresis 1990, 11, 152-155
K. Peisker
Klaus Peisker St.-Elisabeth-Hospital,Halle
Fabric-reinforced ultrathin polyacrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing The suitability of four different fabric materials for the preparation of ultrathin reinforced polyacrylamide gels was investigated. With all fabric-reinforced gels, a good separation of proteins by isoelectric focusing and sodium dodecyl sulfate-electrophoresis could be achieved. Semi-dry electrophoretic blotting of proteins was possible with all types of fabric-reinforced gels. Two polyester fabrics (a net and a fleece) were decidedly superior in handling and dimensional stability on drying to a nylon fabric and another polyester fleece material. Only gels prepared with the former materials withstood further treatment, such as fixation, staining, destaining, and drying. One of the polyester fleece fabrics had poor handling properties and the nylon fabric was unsuitable for direct staining procedures employing concentrated (20 % w/v) trichloroacetic acid as fixative.
1 Introduction The use of ultrathin polyacrylamide gelis, backed to glass or polyester films, has the obvious disadvantage that an electrophoretic transfer is not feasible because both supports do not conduct the electric current. A number of publications 1 1-51 have recently shown that this disadvantage can be overcome by using fabric-reinforced gels. In this report different fabrics are compared with emphasis on ultrathin gels, which are known to offer a number of advantages 161. Four different materials were tested for their suitability to prepare 0. I8 and 0.38 mm ultrathin polyacrylamide gels. The influence of different fabrics or fleeces on electrophoretic separation, staining and protein blotting, and their handling properties as well as dimensional stability were evaluated.
2 Materials and methods 2.1 Fabrics The following four fabrics were used: (1) polyester fabric Monodur PES 60 N, kindly provided by Prof. B. J. Radola (Technical University Munich, Freising-Weiihenstephan, FRG); (2) nylon fabric, obtained from Dr. P. KopaEek (Czechoslovak Akademy of Sciences, Ceske Budejovice, CSSR); (3) polyester fleece, supplied by Dr. Starita-Geribaldi (Faculte de MCdecine, Nice, France); (4) polyester fleece ET 250 from the Institute of Textile Technology (Karl-Marx-Stadt, GDR). The fabric is commercially available from VEB Filmfabrik (Wolfen, GDR).
2.2 Reagents
trichloroacetic acid (TCA) were from Merck (Damstadt, FRG), Tris from Fluka (Buchs, Switzerland), the polyester sheets AT 120 were from the VEB Filmfabrik Wolfen (GDR) and the nitrocellulose (NC) blotting membranes from Sartorius (Gottingen, FRG).
2.3 Preparation of fabric-reinforcedgels The fabric-reinforced gels were prepared by a modified flaptechnique 171 (Fig. 1). Two cover sheets (AT 120) were mounted on glass plates by using a few drops of water, uniformly spread with a rubber roller. After drying ofthe surface water, one cover sheet is overlayed with the fabric by simple pressing or glueing (with “Pritt”, Henkel, Dusseldorf, F R G ) of the fabric edges to the upper side of the glass plate. Uncreased fabrics are directly placed on thecover sheet. The spacer strips were fixed with “Pritt”. Creased fabrics were stretched by folding up and glueing the edges of fabric to the upper side of the glass plate. For SDS-electrophoresisthe slot formers were glued onto the cover sheet by using a 0.2 mm thick letter tape (Herlitz AG, Berlin). Onto uncreased fabrics the polymerization solution could be poured directly, followed by slowly lowering the upper glass plate with the cover sheet. In the case of stretched fabrics, it was preferable to pour the polymerization solution on the lower glass plate with the cover sheet and spacers and to lower the upper glass plate with the stretched fabric. The stock solutions had the following compositions [8, 91: Solution A: 20.0 g acrylamide and 0.54 g N,N’-methylenebisacrylamide, dissolved in deionized water in a final volume of 100 mL. Solution B : 193.7 g Tris, 60.0g glycine and 4.0 g SDS dissolved in 800 mL, adjusted with HC1 to pH 8.8, and filled with water to 1000 mL. Solution C : 10 mg acriflavine hydrochloride in 100mL deionized water. Solution D : 1.0 g ammonium persulfate dissolved in water to a final volume of
Acrylamide, N,N‘-methylenebisacrylamide, acriflavine hydrochloride, Gel-Fix sheets, Serva Blue G, sodium dodecyl sulfate (SDS), Servalyt carrier ampholytes T 4-9, N,N,N‘,N‘tetramethylethylenediamine (TEMED) and glycine were from Serva (Heidelberg, FRG), ammonium persulfate and Correspondence:Dr. Klaus Peisker, St.-Elisabeth-Hospital, MauerstraRe 5- 10, DDR-4010 Halle, German Democratic Rjepublic Abbreviations:IEF, isoelectric focusing; NC, nitrocellulose; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TEJMED, N,N,N‘,N‘-tetramethylethylenediamine; Tris, tris(hydroxymethy1)aminoethane i
VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
_____ __ -- - __c -___ -
I
-cover
sheet -. . -
spacer 1 _____ fabric
--_____------ -----________-_____ -----j
-
4
-/
Figure I. Modified “flap-technique” for the preparation of ultrathin fabricreinforced polyacrylamide gels. 01 73-0835/90/0202-0152 $2.50/0
Electrophoresis 1990.11, 152-155
5 mL. Solution E: buffer stock-solution: 24.0 g Tris. 115.2 g glycine, 2.0 g SDS and 0.5 g NaN, supplemented with water to 1000 mL. The polymerization solution for the preparation of isoelectric focusing (IEF) gels consisted of: 3.75 mL solution A, 2.25 mL glycerol (87 %), 1.00 mL Servalyt T 4-9 carrier ampholytes, 1.0 mL solution C, and 7.00 mL water. The solution was deaerated and photopolymeriza-
Ultrathin fabric-reinforced gels
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tion was allowed to proceed under irradiation with a white “neon-light”. The polymerization solution for SDS gels contained 15.0 m L solution A, 10.0 m L solution B, 8.4 m L glycerol (87 %), 100 1L TEMED and 100 pL solution D. Chemical polymerization, without degassing, was carried out for 30 min at room temperature followed by 30 min at 50-60 O C .
Figure 2. Different fabrics used for the preparation of fabric-reinforced gels: (a) polyester fabric, (b) nylon fabric, (c) polyester fleece,(d) polyester fleece ET 250. Same magnification of about 100 times for all fabrics. For origin of materials see Section 2.1.
Figure 3. Semi-dry electrophoretic blotting from different fabric-reinforced SDS-gels. (1) Polyester fabric, (2) nylon fabric, (3) polyester fleece, (4)polyester fleece ET 250. (A) Fabric-reinforced gel stained with Serva Blue G, (B) NC-blot stained with Serva Blue G, (C)residual gel after blotting, stained with Serva Blue G. SDS-electrophoresis of urinary proteins and protein test mixture 4 (Serva), additionally containing hemoglobin, as described in Section 2.4.
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K. Peisker
2.4 SDS electrophoresis The separation was carried out in the FBE Immuno Unit from Pharmacia(Uppsala, Sweden), with power supply Type 30 1 E from VEB Statron (Furstenwalde, GDR). The fabric-reinforced gel, resting on the cover sheet, was mounted on the cooling block on which a few drops of n-nonane were spread to improve adherence and heat exchange. The electrophoresis buffer consisted of 2 x 400 mL of the diluted stock solution E (100 mL solution E plus 700 mL water), without pH adjustment. Thick viscose fleeces were used as bridges between the buffer vessels and the gel. After a prerun for 5 min at 100V, electrophoresis was performed for about 2 h at 250-300 V. The proteins were fixed with 20 % w/v T C A for 30 min and stained with 0.1 % Serva Blue G in water/methanol/acetic acid 5:4: 1 v/v. The same solution was used for destaining. It is recommended to add 2 % glycerol (87 % ) t o the last destaining solution prior to drying.
2.5 Isoelectric focusing The separation was carried out in the FlBE 3000 apparatus (Pharmacia) with power supply Type 4205 from VEB Statron. Electrophoresis strips (Pharmacia), wetted with 0.73 M
Electrophoresis 1990,1I, 152-155
phosphoric acid as anolyte and 0.51 M ethylenediamine as catholyte were used as electrode strips. Following prefocusing for 60 min, the samples were applied using viscose fleece tabs (Forschungsinstitut fur Textiltechnologie, Karl-Marx-Stadt, GDR). After an additional 60 min at 200 V the sample applicators were removed and focusing continued for 2 h, with a stepwise increase of voltage to a final 1200 V and a total of 2200 V x h. The proteins were fixed for 30 min with 20 % w/v TCA and then stained with 0.1 % Serva Blue G in water/ methanol/acetic acid 5:4:1 v/v. The same solution was used for destaining. By adding 1.2 % glycerol (87 %) to the last destaining solution it was possible to dry the 0.18 mm thick fabric-reinforced gels without deformation. Preliminary experiments with a modified method, using colloidal Serva Violet [ l o ] , showed better results than with the Serva Blue method.
2.6 Blotting The protein transfer by use of semi-dry electrophoretic blotting was carried out in a laboratory-made blotting apparatus, with a transfer buffer of the following composition: 70 mL of 1:10 diluted solution E plus 30 mL glycerol (87 %). Transfer conditions were 50-100 mA over 1 h and 15 V. The protein
Figure 4. Ultrathin IEF in fabric-reinforced gels. (a) Polyester fabric 1, (b) polyester fleece ET 250. Separation according to 2.5 with protein test mixture 9 (Serva) and urinary proteins. Staining with Serva Blue 6.
Ultrathin fabric-reinforced gels
Electrophoresis 1990,11, 152-155
blots were stained with 0.1 Serva Blue G and destained in the same solvent as described in Section 2.5.
3 Results and discussion Although the four tested fabrics showed clear differences in their structures (Fig. 2), we did not observe any difference in the quality of separation on IEF, SDS-electrophoresis or blotting (Fig. 3). Direct fixation and staining of the separated proteins in the fabric-reinforced gels was possible, but great difficulties were experienced with nylon and polyester fleece fabric 3. The nylon fabric was degraded in 20 'XI TCA. used for fixation, and in addition astrong background was observed on staining. The thin polyester fleece (material 3) was not able to compensate the swelling and shrinking forces of the gel in different solutions and tended to roll up and to form a wavy surface on drying. This fabric-reinforced gel had to be stretched with the aid of needles before drying. The polyester fabrics 1 and 4 were appreciably better. Owing to the homogeneous structure of polyester fabric 1 the protein bands appeared sharp on IEF (Fig. 4) and a uniform background was found on destaining. The E T 250 fabric (material 4) showed superior handling properties during preparation of ultrathin fabricreinforced gels. The results of testing the four different fabrics for the preparation of ultrathin fabric-reinforced gels and for blotting may be summarized asfollows: (i)The ET 250 and the polyester fabric (material 1) were decidedly better in handling and dimensional stability on drying than the nylon fabric or polyester material 3. (ii) Despite great differences in structure all 4 materials proved suitable as gel-stabilizing supports for IEF, SDS-electrophoresis and blotting. (iii) Only gels reinforced with fabric 1 or 4 withstood further treatment, such as fixation, staining, destaining, and drying. The nylon fabric deteriorated during fixation in concentrated TCA and also showed an unacceptable background staining. Material 3 had a nonuniform background following staining and poor dimensional stability on drying. (iv) The ET 250 material was the only fabric with a wick-effect, allowing the reinforced gels to be prepared without preconditioning (e.g. washing). The good
155
dimensional stability of this material facilitated the preparation of ultrathin fabric-reinforced polyacrylamide gels for IEF and SDS-electrophoresis. (v) The modified flap-technique is well suited to prepare air-bubble-free, ultrathin fabric-reinforced gels. Still better gels were obtained using a method of Kinzkofer-Peresch et al. 151or using washed (0.01 o/o of anonionic detergent, Triton X- 100)and dried fabrics. In both cases moistening with the gel solution was improved.
4 Concluding remarks A comparison offour different fabrics has shown that all were suitable for the preparation of fabric reinforced gels. Two polyester fabrics, a net and a fleece, proved superior with regard to handling properties during gel preparation, dimensional stability of the fabric-reinforced gel, stability on fixation and staining. Received January 10, 1989; in revised for August 22, 1989
5 References 111 Nishizawa, H., Murakami, A., Hayashi, N., Iida, M. and Abe, Y., Electrophoresis 1985,6, 349-350. (21 Kinzkofer, A. and Radola, B. J., in: Radola, B. J . (Ed.), Elektrophorese Forum '86, Technische Universitat, Munchen 1986, pp. 253-257. 131 Saravis, C. A., Cook, R. B.. Polvino, W. J. and Sampson, C. E., Electrophoresis 1983,4, 367. 141 Starita-Geribaidi, M., Guidicelli, J. and Sudaka, P.. Electrophoresis 1988,9,234-236. 1.51 Kinzkofer-Peresch, A., Patestos,N. P.,Fauth, M.,KOgel. F.,Zok. R. and Radola, B. J., Electrophoresis 1988,9,497-5 11. [61 Gorg, A,, Postel, W. and Westermeier, R., GIT-Labormedizln 1979. 2, 32-40. 171 Radola, B. J., Electrophoresis 1980,1,43-56. 181 Peisker, K., Z . Med. Lab. Diagn. 1985, 26, 28-33. 191 Peisker, K., Z . Med. Lab. Diagn. 1987,28,38-42. 101 Patestos, N. P., Fauth, M. and Radola, B. J., Electrophoresis 1998.9, * 488-496.
Electrophoresis 1990, 11, 152-155
K. Peisker
Klaus Peisker St.-Elisabeth-Hospital,Halle
Fabric-reinforced ultrathin polyacrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing The suitability of four different fabric materials for the preparation of ultrathin reinforced polyacrylamide gels was investigated. With all fabric-reinforced gels, a good separation of proteins by isoelectric focusing and sodium dodecyl sulfate-electrophoresis could be achieved. Semi-dry electrophoretic blotting of proteins was possible with all types of fabric-reinforced gels. Two polyester fabrics (a net and a fleece) were decidedly superior in handling and dimensional stability on drying to a nylon fabric and another polyester fleece material. Only gels prepared with the former materials withstood further treatment, such as fixation, staining, destaining, and drying. One of the polyester fleece fabrics had poor handling properties and the nylon fabric was unsuitable for direct staining procedures employing concentrated (20 % w/v) trichloroacetic acid as fixative.
1 Introduction The use of ultrathin polyacrylamide gelis, backed to glass or polyester films, has the obvious disadvantage that an electrophoretic transfer is not feasible because both supports do not conduct the electric current. A number of publications 1 1-51 have recently shown that this disadvantage can be overcome by using fabric-reinforced gels. In this report different fabrics are compared with emphasis on ultrathin gels, which are known to offer a number of advantages 161. Four different materials were tested for their suitability to prepare 0. I8 and 0.38 mm ultrathin polyacrylamide gels. The influence of different fabrics or fleeces on electrophoretic separation, staining and protein blotting, and their handling properties as well as dimensional stability were evaluated.
2 Materials and methods 2.1 Fabrics The following four fabrics were used: (1) polyester fabric Monodur PES 60 N, kindly provided by Prof. B. J. Radola (Technical University Munich, Freising-Weiihenstephan, FRG); (2) nylon fabric, obtained from Dr. P. KopaEek (Czechoslovak Akademy of Sciences, Ceske Budejovice, CSSR); (3) polyester fleece, supplied by Dr. Starita-Geribaldi (Faculte de MCdecine, Nice, France); (4) polyester fleece ET 250 from the Institute of Textile Technology (Karl-Marx-Stadt, GDR). The fabric is commercially available from VEB Filmfabrik (Wolfen, GDR).
2.2 Reagents
trichloroacetic acid (TCA) were from Merck (Damstadt, FRG), Tris from Fluka (Buchs, Switzerland), the polyester sheets AT 120 were from the VEB Filmfabrik Wolfen (GDR) and the nitrocellulose (NC) blotting membranes from Sartorius (Gottingen, FRG).
2.3 Preparation of fabric-reinforcedgels The fabric-reinforced gels were prepared by a modified flaptechnique 171 (Fig. 1). Two cover sheets (AT 120) were mounted on glass plates by using a few drops of water, uniformly spread with a rubber roller. After drying ofthe surface water, one cover sheet is overlayed with the fabric by simple pressing or glueing (with “Pritt”, Henkel, Dusseldorf, F R G ) of the fabric edges to the upper side of the glass plate. Uncreased fabrics are directly placed on thecover sheet. The spacer strips were fixed with “Pritt”. Creased fabrics were stretched by folding up and glueing the edges of fabric to the upper side of the glass plate. For SDS-electrophoresisthe slot formers were glued onto the cover sheet by using a 0.2 mm thick letter tape (Herlitz AG, Berlin). Onto uncreased fabrics the polymerization solution could be poured directly, followed by slowly lowering the upper glass plate with the cover sheet. In the case of stretched fabrics, it was preferable to pour the polymerization solution on the lower glass plate with the cover sheet and spacers and to lower the upper glass plate with the stretched fabric. The stock solutions had the following compositions [8, 91: Solution A: 20.0 g acrylamide and 0.54 g N,N’-methylenebisacrylamide, dissolved in deionized water in a final volume of 100 mL. Solution B : 193.7 g Tris, 60.0g glycine and 4.0 g SDS dissolved in 800 mL, adjusted with HC1 to pH 8.8, and filled with water to 1000 mL. Solution C : 10 mg acriflavine hydrochloride in 100mL deionized water. Solution D : 1.0 g ammonium persulfate dissolved in water to a final volume of
Acrylamide, N,N‘-methylenebisacrylamide, acriflavine hydrochloride, Gel-Fix sheets, Serva Blue G, sodium dodecyl sulfate (SDS), Servalyt carrier ampholytes T 4-9, N,N,N‘,N‘tetramethylethylenediamine (TEMED) and glycine were from Serva (Heidelberg, FRG), ammonium persulfate and Correspondence:Dr. Klaus Peisker, St.-Elisabeth-Hospital, MauerstraRe 5- 10, DDR-4010 Halle, German Democratic Rjepublic Abbreviations:IEF, isoelectric focusing; NC, nitrocellulose; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TEJMED, N,N,N‘,N‘-tetramethylethylenediamine; Tris, tris(hydroxymethy1)aminoethane i
VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
_____ __ -- - __c -___ -
I
-cover
sheet -. . -
spacer 1 _____ fabric
--_____------ -----________-_____ -----j
-
4
-/
Figure I. Modified “flap-technique” for the preparation of ultrathin fabricreinforced polyacrylamide gels. 01 73-0835/90/0202-0152 $2.50/0
Electrophoresis 1990.11, 152-155
5 mL. Solution E: buffer stock-solution: 24.0 g Tris. 115.2 g glycine, 2.0 g SDS and 0.5 g NaN, supplemented with water to 1000 mL. The polymerization solution for the preparation of isoelectric focusing (IEF) gels consisted of: 3.75 mL solution A, 2.25 mL glycerol (87 %), 1.00 mL Servalyt T 4-9 carrier ampholytes, 1.0 mL solution C, and 7.00 mL water. The solution was deaerated and photopolymeriza-
Ultrathin fabric-reinforced gels
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tion was allowed to proceed under irradiation with a white “neon-light”. The polymerization solution for SDS gels contained 15.0 m L solution A, 10.0 m L solution B, 8.4 m L glycerol (87 %), 100 1L TEMED and 100 pL solution D. Chemical polymerization, without degassing, was carried out for 30 min at room temperature followed by 30 min at 50-60 O C .
Figure 2. Different fabrics used for the preparation of fabric-reinforced gels: (a) polyester fabric, (b) nylon fabric, (c) polyester fleece,(d) polyester fleece ET 250. Same magnification of about 100 times for all fabrics. For origin of materials see Section 2.1.
Figure 3. Semi-dry electrophoretic blotting from different fabric-reinforced SDS-gels. (1) Polyester fabric, (2) nylon fabric, (3) polyester fleece, (4)polyester fleece ET 250. (A) Fabric-reinforced gel stained with Serva Blue G, (B) NC-blot stained with Serva Blue G, (C)residual gel after blotting, stained with Serva Blue G. SDS-electrophoresis of urinary proteins and protein test mixture 4 (Serva), additionally containing hemoglobin, as described in Section 2.4.
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K. Peisker
2.4 SDS electrophoresis The separation was carried out in the FBE Immuno Unit from Pharmacia(Uppsala, Sweden), with power supply Type 30 1 E from VEB Statron (Furstenwalde, GDR). The fabric-reinforced gel, resting on the cover sheet, was mounted on the cooling block on which a few drops of n-nonane were spread to improve adherence and heat exchange. The electrophoresis buffer consisted of 2 x 400 mL of the diluted stock solution E (100 mL solution E plus 700 mL water), without pH adjustment. Thick viscose fleeces were used as bridges between the buffer vessels and the gel. After a prerun for 5 min at 100V, electrophoresis was performed for about 2 h at 250-300 V. The proteins were fixed with 20 % w/v T C A for 30 min and stained with 0.1 % Serva Blue G in water/methanol/acetic acid 5:4: 1 v/v. The same solution was used for destaining. It is recommended to add 2 % glycerol (87 % ) t o the last destaining solution prior to drying.
2.5 Isoelectric focusing The separation was carried out in the FlBE 3000 apparatus (Pharmacia) with power supply Type 4205 from VEB Statron. Electrophoresis strips (Pharmacia), wetted with 0.73 M
Electrophoresis 1990,1I, 152-155
phosphoric acid as anolyte and 0.51 M ethylenediamine as catholyte were used as electrode strips. Following prefocusing for 60 min, the samples were applied using viscose fleece tabs (Forschungsinstitut fur Textiltechnologie, Karl-Marx-Stadt, GDR). After an additional 60 min at 200 V the sample applicators were removed and focusing continued for 2 h, with a stepwise increase of voltage to a final 1200 V and a total of 2200 V x h. The proteins were fixed for 30 min with 20 % w/v TCA and then stained with 0.1 % Serva Blue G in water/ methanol/acetic acid 5:4:1 v/v. The same solution was used for destaining. By adding 1.2 % glycerol (87 %) to the last destaining solution it was possible to dry the 0.18 mm thick fabric-reinforced gels without deformation. Preliminary experiments with a modified method, using colloidal Serva Violet [ l o ] , showed better results than with the Serva Blue method.
2.6 Blotting The protein transfer by use of semi-dry electrophoretic blotting was carried out in a laboratory-made blotting apparatus, with a transfer buffer of the following composition: 70 mL of 1:10 diluted solution E plus 30 mL glycerol (87 %). Transfer conditions were 50-100 mA over 1 h and 15 V. The protein
Figure 4. Ultrathin IEF in fabric-reinforced gels. (a) Polyester fabric 1, (b) polyester fleece ET 250. Separation according to 2.5 with protein test mixture 9 (Serva) and urinary proteins. Staining with Serva Blue 6.
Ultrathin fabric-reinforced gels
Electrophoresis 1990,11, 152-155
blots were stained with 0.1 Serva Blue G and destained in the same solvent as described in Section 2.5.
3 Results and discussion Although the four tested fabrics showed clear differences in their structures (Fig. 2), we did not observe any difference in the quality of separation on IEF, SDS-electrophoresis or blotting (Fig. 3). Direct fixation and staining of the separated proteins in the fabric-reinforced gels was possible, but great difficulties were experienced with nylon and polyester fleece fabric 3. The nylon fabric was degraded in 20 'XI TCA. used for fixation, and in addition astrong background was observed on staining. The thin polyester fleece (material 3) was not able to compensate the swelling and shrinking forces of the gel in different solutions and tended to roll up and to form a wavy surface on drying. This fabric-reinforced gel had to be stretched with the aid of needles before drying. The polyester fabrics 1 and 4 were appreciably better. Owing to the homogeneous structure of polyester fabric 1 the protein bands appeared sharp on IEF (Fig. 4) and a uniform background was found on destaining. The E T 250 fabric (material 4) showed superior handling properties during preparation of ultrathin fabricreinforced gels. The results of testing the four different fabrics for the preparation of ultrathin fabric-reinforced gels and for blotting may be summarized asfollows: (i)The ET 250 and the polyester fabric (material 1) were decidedly better in handling and dimensional stability on drying than the nylon fabric or polyester material 3. (ii) Despite great differences in structure all 4 materials proved suitable as gel-stabilizing supports for IEF, SDS-electrophoresis and blotting. (iii) Only gels reinforced with fabric 1 or 4 withstood further treatment, such as fixation, staining, destaining, and drying. The nylon fabric deteriorated during fixation in concentrated TCA and also showed an unacceptable background staining. Material 3 had a nonuniform background following staining and poor dimensional stability on drying. (iv) The ET 250 material was the only fabric with a wick-effect, allowing the reinforced gels to be prepared without preconditioning (e.g. washing). The good
155
dimensional stability of this material facilitated the preparation of ultrathin fabric-reinforced polyacrylamide gels for IEF and SDS-electrophoresis. (v) The modified flap-technique is well suited to prepare air-bubble-free, ultrathin fabric-reinforced gels. Still better gels were obtained using a method of Kinzkofer-Peresch et al. 151or using washed (0.01 o/o of anonionic detergent, Triton X- 100)and dried fabrics. In both cases moistening with the gel solution was improved.
4 Concluding remarks A comparison offour different fabrics has shown that all were suitable for the preparation of fabric reinforced gels. Two polyester fabrics, a net and a fleece, proved superior with regard to handling properties during gel preparation, dimensional stability of the fabric-reinforced gel, stability on fixation and staining. Received January 10, 1989; in revised for August 22, 1989
5 References 111 Nishizawa, H., Murakami, A., Hayashi, N., Iida, M. and Abe, Y., Electrophoresis 1985,6, 349-350. (21 Kinzkofer, A. and Radola, B. J., in: Radola, B. J . (Ed.), Elektrophorese Forum '86, Technische Universitat, Munchen 1986, pp. 253-257. 131 Saravis, C. A., Cook, R. B.. Polvino, W. J. and Sampson, C. E., Electrophoresis 1983,4, 367. 141 Starita-Geribaidi, M., Guidicelli, J. and Sudaka, P.. Electrophoresis 1988,9,234-236. 1.51 Kinzkofer-Peresch, A., Patestos,N. P.,Fauth, M.,KOgel. F.,Zok. R. and Radola, B. J., Electrophoresis 1988,9,497-5 11. [61 Gorg, A,, Postel, W. and Westermeier, R., GIT-Labormedizln 1979. 2, 32-40. 171 Radola, B. J., Electrophoresis 1980,1,43-56. 181 Peisker, K., Z . Med. Lab. Diagn. 1985, 26, 28-33. 191 Peisker, K., Z . Med. Lab. Diagn. 1987,28,38-42. 101 Patestos, N. P., Fauth, M. and Radola, B. J., Electrophoresis 1998.9, * 488-496.
