Isoelectric Focusing In Ultrathin Gels Research Articles

Investigation of CSF proteins using high voltage isoelectric focusing in ultrathin gels and enzyme amplified immuno sandwich assay.

Investigation of CSF proteins using high voltage isoelectric focusing in ultrathin gels and enzyme amplified immuno sandwich assay.

Microheterogeneity of human erythrocyte pyruvate kinase: Application of immunological visualization techniques after isoelectric focusing in ultrathin gels

Immunological methods for protein visualization afford high sensitivity, good specificity and resolution. We used the indirect immunoassay with peroxidase- and 125I-labelled antibodies to investigate the microheterogeneity of pyruvate kinase from human red blood cells and can show an enzyme pattern with more than 10 single bands. Different methods were tested as to their applicability for protein fixation and subsequent immunological visualization in polyacrylamide gels. Besides the Western blot, immunofixation and ethanolic fixation can be recommended as fixation techniques, especially after isoelectric focusing in ultrathin gels.

High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: a new method for analysis of cerebrospinal fluid proteins.

A procedure using high-voltage isoelectric focusing (IF) in ultrathin (02. mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000-3000 V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with "normal" CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum evaporation technique before IF.