Concluding Remarks

Gradient gel electrophoresis has may advantages over conventional gel electrophoresis. Gradient range and course can be adapted to any individual separation problem, and protein bands are much sharper than in Cellogel or starch gel electrophoresis. So far, for example, more than 20 enzyme bands of an enzyme system such as plant acid phosphatase have been clearly resolved and genetically interpreted, and crude enzyme extracts can be used as the enzyme source, provided a specific detection (staining) system is available. The disadvantages are few compared to conventional gel electrophoresis, such as availability of gradient gel, a load of two to three times more enzyme activity per cm2 of gel cross-section as compared to starch gels, and the exclusive migration of proteins towards the cathode (anode), whereas in Cellogel and starch gel electrophoresis both cathodi-cally and anodically migrating proteins can be detected within the matrix.

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Figure 16 Separation of urinary proteins by macro SDS PA gradient gel electrophoresis. Gel: 4-20% T. Running conditions: 3 h at 350 V, 50 mA. Samples: up to 50 ^L urine. (A) and (B) show urine samples from paediatric patients with pyelonephritis at various stages of follow-up. Series A: 1, acute phase: 2, and 3, urine taken at weekly intervals; 4, reinfection (acute phase); 5,1 week follow-up. Series B: 1, acute phase; 2-4, follow-up at weekly intervals (note blood contamination in 2 and 3, a- and ^-globin chains at 16 kDa), 5, reinfection, 6, and 7, weekly follow-up. Black dot and vertical arrow on the right side of the gel represent the application point and migration direction, respectively. # Gel polarity. Alb., albumin. Reproduced with permission from Bianchi-Bosisio et al. (1991).

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Figure 16 Separation of urinary proteins by macro SDS PA gradient gel electrophoresis. Gel: 4-20% T. Running conditions: 3 h at 350 V, 50 mA. Samples: up to 50 ^L urine. (A) and (B) show urine samples from paediatric patients with pyelonephritis at various stages of follow-up. Series A: 1, acute phase: 2, and 3, urine taken at weekly intervals; 4, reinfection (acute phase); 5,1 week follow-up. Series B: 1, acute phase; 2-4, follow-up at weekly intervals (note blood contamination in 2 and 3, a- and ^-globin chains at 16 kDa), 5, reinfection, 6, and 7, weekly follow-up. Black dot and vertical arrow on the right side of the gel represent the application point and migration direction, respectively. # Gel polarity. Alb., albumin. Reproduced with permission from Bianchi-Bosisio et al. (1991).

In addition to pure PA gels, matrices of mixed polymers can be used for porosity gradients. However, this possibility has been rarely used, although it could extend the separation possibilities.

PA gradients are widely used to determine the molecular mass of SDS-denatured proteins, because this method offers a larger separation range and a much better resolution of protein bands. However, native, time-dependent PA gradient gel electrophoresis has much more possibilities to offer, such as differentiation between size and charge isomers, determination of the molecular size of native proteins and (iso)en-zymes (Mr, RS), estimation of the molecular excentric-ity (f/fo), and calculation of the net negative charge at a given pH value. Using these possibilities the evolution of homologous proteins in related animal and plant species can be studied as well as the net charge of isozymes from different cells compartments.

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