N-N' diallyltartardiamide (DATD) as a cross-linking agent for polyacrylamide gel disc electrophoresis of human serum proteins.
Polyacrylamide gel disc electrophoresis is a sensitive, versatile and popular analytical method for resolving mixtures of proteins in biological fluids. The conventional method involves the use of N-N' methylenebisacrylamide (BIS) as a crosslinking agent to give rigidity and to form the acrylamide gel. The pore size of the gel is determined mainly by the concentration of acrylamide monomer and to a lesser extent by the cross-linking agent. Anker described the use of another comonomer or cross-linking agent N-N' Diallyltartardiamide (DATD) in casting acrylamide gels. This compound is structurally similar to BIS but has the advantage of possessing cis-diol groups which can be cleaved by periodic acid. Thus, in contrast to BIS gels, DATD gels can be solubilized and are called reversible gels. This property is very useful in eluting and measuring radio-labelled materials. Another agent with DATD like properties of forming reversible gels is Ethylene diacrylate. We have been able to obtain only four pertinent studies of the use of DATD in polyacrylamide gels.,,, Besides their solubility in periodic acid, these gels have the advantages of being unusually transparent, elastic and of having better binding to glass. They show swelling on storage,, Studies describing the use of DATD gels have been restricted to the analyses of pure proteins. There are no reports of analysis of mixtures. We describe here our observations on 50 duplicate disc electrophoretic separations of human serum proteins in conventional BIS and the newer DATD linked acrylamide gels.
Sera were obtained from 50 normal healthy adults in the age range of 20 to 40 years. They included 20 females and 30 males. Care was taken to study fresh sera. Turbid and hemolytic sera were excluded.
Disc electrophoresis was carried out as described by Davis. All reagents were of analytical grade quality. Both BIS and DATD were used in a concentration of 0.25 g/dL. Each sample was run in duplicate in a BIS and in a DATD cross-linked gel. The serum innoculum was 10µl and the marker, one per cent aqueous bromophenol blue. The buffer was Tris (50 mmol/L) and Glycine (380 mmol/L) pH 8.3. Electrophoresis was carried out at 4°C with a constant current of 3 mA per gel. This was discontinued when the marker dye reached the end of the gel. The time of electrophoresis was recorded.
Protein staining: The gels were removed carefully and stained with one per cent Amido Black in seven per cent acetic acid for five minutes. Destaining was carried out with seven per cent acetic acid for 24 to 96 hours.
Recording of results: The individual components were identified according of the scheme of Davis. The markers were albumin, (fastest moving and most prominent band), transferrin (prominent band in the middle of the gel) and betalipoprotein (slowest prominent band). The protein bands in the post-albumin and the post-transferrin regions were noted and the number of bands and distance of migration recorded. The E, values were calculated by using the formula.
Ef = Distance travelled by protein
Distance travelled by albumin
The migration velocity (M) is preferred and was calculated by the formula:
The swelling of the gels was assessed by measuring the diameters immediately after removal from the tube and 15 days later.
The figure illustrates the results of protein staining of a serum electrophoretic separation in a DATD and in a BIS linked gel. DATD gels were soft, elastic and quite transparent. They showed a swelling of 30 per cent after storage for 15 days. Resolution was better in DATD gels because of the sparklingly clear background due to better removal of dye in the course of decolourisation. Protein bands in the DATD gels could be identified easily (See Fig. 1 on page ....) but there was some difficulty with definition of bands in the BIS-linked gels. DATD gels, however, showed a diffusion of bands and some fading at 15 days which was not noted in the BIS gels. Polymerization of DATD gels took 90 minutes in comparison with 45 minutes for BIS gels.
[Table - 1] compares the number of protein bands obtained in the duplicate analyses of electrophoretic separations of 50 normal human sera using either DATD or BIS. Identical results were obtained with 30 sera. The details of differences seen in the remaining 20 sera are shown in [Table - 2]. DATD gels resolved more protein bands in 11 sera and BIS gave more bands in nine sera. The 'M' values of some proteins in DATD and BIS-linked gels are shown in [Table 3]. The migration velocities in the DAM gels are much greater.
The results of the present study indicate the suitability of DATD as a cross-linking agent for polyacrylamide gel electrophoresis of mixtures like human serum proteins. This agent has an advantage over BIS because of the remarkable clarity of gels between protein bands. The low background stain of the DATD gels was an advantage when studying the closely set components of the post-transferrin region. In terms of the number of bands obtained, DATD gels were also better than BIS gels.
The migration velocity (M) in DATD gels was greater than in BIS gels [Table 3]. (We have preferred M to E because it is a more specific parameter). Baumann and Chrombach have related this to the larger pore-size of such gels. These authors used highly cross-linked DATD gels for studies of a variety of pure proteins. They described the advantages of DATD gels to be improved stability, better resolution, better adherence to glass and greater transparency. The swelling of DATD gels on storage after electrophoresis was considerable. This, however, did not interfere or distort the banding pattern of human serum protein separations. Comparing the cost of DATD and BIS linked gels, the DATD gels worked out to be twenty times costlier than the BIS linked gels.
Of course, the main advantage of DATD is that the gel can be solubilized by periodic acid. This makes it possible to elute components more elegantly and completely than from BIS gels. Even if solubility of the gel is not the objective of this study there is a strong case for the use of DATD gels. This is because of its clarity, better resolution and handling characteristics.