Isoelectric focusing ultrathin-layer

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Fig. 5. Isoelectric focusing (pH gradient 3-10) of Fraction A and Fraction B in ultrathin polyacrylamide layers. 5 pg of fractions were applied. Activity detection with ruthenium red (left) and with Ostazin Brilliant Red/D-galacturonan DP 10 agar print (right).

Ultrathin-layer isoelectric focusing in polyacrylamide gels on polyester films was performed as described (Radola, 1980). Polygalacturonase activity was detected by the print technique with a dyed substrate (Ostazin Brilliant Red-D-galacturonan DP 10) (Markovic et al., 1992) or by the print technique with colouress D-galacturonan DP 10 dyed additionally with ruthenium red (Sigma, Germany). 

Fig. 3. Isoelectric focusing in ultrathin polyacrylamide layers (pH gradient 3 -10) of multiple forms of polygalacturonase produced by Candida boidinii under different cultivation conditions a - pectin, pH 3.51 b - pectin, pH 5.49 c -pectin, pH 7.01 d - 20% of D-galactopyranuronic acid in pectin e - pectate. A - Activity detection with print technique on colouress D-galacturonan DP 10 dyed additionally with ruthenium red ( both exo- and polygalacturonases) and B - activity detection with Ostazin Brilliant Red/D-galacturonan DP 10 agar print (polygalacturonases).

Rehydratable Polyacrylamide Gels for Ultrathin-Layer Isoelectric Focusing... 

Figure 3. The effect of separators on ultrathin-layer isoelectric focusing in rehydratable gels. Gels (120 jjim) containing 10% Dextran 35 plus 3% glycerol were rehydrated with 3% Servalyt pH 4-6 plus 5%, glycerol with the indicated concentration of different separators. On the left, a mixture of four marker proteins MYH-horse myoglobin, CAR-carbonic anhydrase, LAC-(3-lactoglobulin,...

Standardization in Isoelectric Focusing on Ultrathin-Layer Rehydratable Polyacrylamide Gels... 

Berg, W., Gutschker-Gdaniec, G., and Schauer, R., 1985, Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing. Anal. Biochem. 145 339-342. 

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