A More Universal And Stable Method For Lactate Ddehydrogenase Isoenzymes Test

Objective: Establish a more stable lactate dehydrogenase zymography technology using for different species.Methods: Design and assemble an automatic constant temperature control system to control the running buffer temperature constantly,and use the ice water bath to set a series of electrophoresis temperatures,so as to investigate the effects of ice-water bath and room temperature electrophoresis on lactate dehydrogenase zymography technology.Set a series of pH values of running buffer through a pH meter to investigate the influence of running buffer pH on lactate dehydrogenase zymography technology and select an optimal one.Compare the database prediction data and experimental data to find the possible reason for those species differences.Use different running buffer to investigate the influence of running buffer type on lactate dehydrogenase zymography technology.Results: There were 5 bands in the low-temperature group and 4 bands in the normal temperature group,cause its LDH5 was inactivated.In the constant temperature(10 ?)group,the temperature fluctuation was within 2 ? and all LDH isoenzymes were detected and separated well without being trapped into the channel,while in the normal temperature group,the temperature fluctuation was over 10?,and the LDH5 was inactivated and the LDH4 was trapped into the channel.In the constant temperature(10 ?)group,all LDH isoenzymes were detected and separated well without being trapped into the channel,while in other constant temperature groups(15 ?,20 ?,25 ?)there were bands missing and trapping in some degree.An obvious difference in the mobility of LDH isoenzymes when the temperature of running buffer changed by 5 ? and the LDH5 expressed a trend of degradation with the increase of temperature.The enzyme mobility varied with running buffer Ph,when the pH was 8.3 or 8.6,the LDH isoenzymes had the largest gap.LDH3 and LDH4 were equally distributed at both sides of the channel with no retention.As the pH of running buffer increased,the trapping of cathode bands decreased;and as the pH of running buffer decreased,the trapping of anode bands decreased.The enzyme activity became weaker and weaker as the actual pH deviated further and further from the primary pH of TBE buffer.The mobility of rat and human LDH isoenzymes are consistent while that of mouse was totally different.The prediction data from NCBI Protein Database and the experimental data are consistent,that is the same isoenzyme of human and rat sources had the same mobility and was at the same level in the zymogram,while the same isozyme in mouse was significantly different and at different levels in the zymogram which conformed to the predicted PI of these species.In the TBE buffer group,the heating efficiency and running buffer current are low and the constant temperature system can control its temperature constantly,while in the barbital buffer group isn't that case.This improved method can be used for different species and sample types and can fulfill the quantitative analysis.Conclusion: The temperature of running buffer is crucial for the stability of the LDH zymography and 10? is the best.The pH of running buffer significantly influenced the enzyme mobility,different species should match with different running buffer pH as well as the actual pH should be set near the primary pH.When the cathode bands are trapped,raising the pH slightly can improve the results,when the anode bands are trapped,choosing the opposite.The best choice of a marker should be the one from the same species with the studies and can display five bands in its zymogram.Setting the mixed sample from different tissues of the same species as the marker is acceptable.When the LDH zymography comparison of the marker species and study species is known,even the marker is of different species it can be used.The polyacrylamide gel electrophoresis has some limitations in LDH zymography technology while agarose gel electrophoresis is better.The running buffer should be set as the one which heating ratio is low and its primary pH match with its actual pH.