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P.18. Carbamylation trains standards for two-dimensional electrophoresis
P.18. C a r b a m y l a t i o n electrophoresis
trains
standards
for
two-dimensional
J. Lizana 1 and K.-E. Johansson 2. 1pharmacia AB, S-751 82 UPPSALA, Sweden. 2The National Veterinary Institute, Box 7073, S-750 07 UPPSALA, Sweden. The rapid acceptance of 2-D electrophoresis in the various areas of bioscience research poses new demands on the precision and accuracy of standardization for intra- and inter-laboratory comparisons of 2-D maps. In a 2-D gel the positions of the polypeptide spots are best defined using a system of MW and pI coordinates. Molecular weight standards for the SDS-PAGE dimension are readily available. However the standardization of the locations of the spots in the first IEF dimension are more difficult because the presence of high concentrations of urea can make the measurement of the pH gradient very inaccurate, since the pI of some proteins is unpredictably displaced. Nevertheless, several approaches have been used with variable success to standardize the first dimension of 2-D electrophoresis: 1) Measurement of the pH in water extracts of 5 mm slices from the gel or direct used of a surface electrode; 2) Internal standards in the sample, e.g. ribosomal proteins $1,L7 and L12 present in all samples of E.coli samples; 3) Mixture of selected purified proteins of known MW and pI; 4) Use of fluorescent dansylated proteins; 5) Carbamylated proteins, also called "carbamylation trains". It is on these "carbamylation trains" that the new Carbamylyte (Pharmacia AB) standards are based. Carbamylyte standards consist of 3 separate, highly purified carbamylated protein mixtures: carbonic anhydrase(CA), creatine phosphokinase(CPK) and glyceraldehyde-3-phospphate dehydrogenase(GAPDH). Each Carbamylyte product gives a series of spots of known MW across a defined pH range(Table 1). Each of the spots can be identified by counting from the native spot, which is assigned number 0, towards the acidic region(usually the left side of the gel). This negative numbering system introduced by Anderson and Hickman (Anal, Biochem. 93:312,1979) , is based on the fact that each carbamylation step introduces a negative charge into the protein. A Carbamylyte thus extend from 0 to -19 in the case of CA Carbamylyte.
47
Table 1. Some properties of Carbamylyte TM Protein
Subunit MW
Native pI
No.of spots
Apparent range of use (pI)
CA CPK GADPH
30,000 40,000 36,000
5.8 7.0 8.5
20 34-36 34
4.8 -6.7 4.9 - 7.1 4.7 - 8.3.
In this paper, Carbamylyte T M has been used for two purposes. First, it was used to optimize the shape of the pH gradient for separation of E. coli proteins. With Carbamylyte as mode1 samples, the combination of ampholytes giving optimal separation for the E. coli sample can thus be pinpointed and optimized, thus saving precious sample. The use of Carbamylyte and MW markers also allowed the introduction of a coordinate system, which greatly facilitated the identification of protein spots and made comparisons of 2-D maps much easier.
48
trains
standards
for
two-dimensional
J. Lizana 1 and K.-E. Johansson 2. 1pharmacia AB, S-751 82 UPPSALA, Sweden. 2The National Veterinary Institute, Box 7073, S-750 07 UPPSALA, Sweden. The rapid acceptance of 2-D electrophoresis in the various areas of bioscience research poses new demands on the precision and accuracy of standardization for intra- and inter-laboratory comparisons of 2-D maps. In a 2-D gel the positions of the polypeptide spots are best defined using a system of MW and pI coordinates. Molecular weight standards for the SDS-PAGE dimension are readily available. However the standardization of the locations of the spots in the first IEF dimension are more difficult because the presence of high concentrations of urea can make the measurement of the pH gradient very inaccurate, since the pI of some proteins is unpredictably displaced. Nevertheless, several approaches have been used with variable success to standardize the first dimension of 2-D electrophoresis: 1) Measurement of the pH in water extracts of 5 mm slices from the gel or direct used of a surface electrode; 2) Internal standards in the sample, e.g. ribosomal proteins $1,L7 and L12 present in all samples of E.coli samples; 3) Mixture of selected purified proteins of known MW and pI; 4) Use of fluorescent dansylated proteins; 5) Carbamylated proteins, also called "carbamylation trains". It is on these "carbamylation trains" that the new Carbamylyte (Pharmacia AB) standards are based. Carbamylyte standards consist of 3 separate, highly purified carbamylated protein mixtures: carbonic anhydrase(CA), creatine phosphokinase(CPK) and glyceraldehyde-3-phospphate dehydrogenase(GAPDH). Each Carbamylyte product gives a series of spots of known MW across a defined pH range(Table 1). Each of the spots can be identified by counting from the native spot, which is assigned number 0, towards the acidic region(usually the left side of the gel). This negative numbering system introduced by Anderson and Hickman (Anal, Biochem. 93:312,1979) , is based on the fact that each carbamylation step introduces a negative charge into the protein. A Carbamylyte thus extend from 0 to -19 in the case of CA Carbamylyte.
47
Table 1. Some properties of Carbamylyte TM Protein
Subunit MW
Native pI
No.of spots
Apparent range of use (pI)
CA CPK GADPH
30,000 40,000 36,000
5.8 7.0 8.5
20 34-36 34
4.8 -6.7 4.9 - 7.1 4.7 - 8.3.
In this paper, Carbamylyte T M has been used for two purposes. First, it was used to optimize the shape of the pH gradient for separation of E. coli proteins. With Carbamylyte as mode1 samples, the combination of ampholytes giving optimal separation for the E. coli sample can thus be pinpointed and optimized, thus saving precious sample. The use of Carbamylyte and MW markers also allowed the introduction of a coordinate system, which greatly facilitated the identification of protein spots and made comparisons of 2-D maps much easier.
48