This method provides a better understanding of the structural organizations of glycogen particles. It is an important issue because the population of the Glucan chains, the so-called chain length distribution, determines the physical chemical properties of the glycogen particles. Such as water solubility.
One major advantage of this technique is that fluorescence is constant regardless of Glucan chain length. There is only one APTS molecule bound by Glucan. Thus, fluorescence intensity is directly proportional to the number of Glucan chains.
The field for assisted capillary electrophoresis is suitable for solving the medical mechanism of glycogen metabolizing enzymes. For instance, we can determine the degree of polymerization of Glucan chains transferred by branching enzymes onto accepted Glucan. The reaction must be performed in conditions.
Therefore, regions must be protected from moisture. Mix 200 microliters of 0.5-2 milligrams per milliliter of purified glycogen with 200 microliters of 100 acetate buffer of pH 4.8. Add 2 microliters of Isoamylase, and 1.5 microliters of Pullulanase.
Mix gently by pipetting up and down. Then incubate at 42 degrees Celsius for 16 hours in a 1.5 milliliter tube. Stop the reactions by incubating at 95 degrees Celsius for 5 minutes.
Centrifuge at 16, 100 times G'for 5 minutes at room temperature to pellet and remove any insoluble material. Remove the super maintenance with a pipette and transfer them to new annotated tubes. After adding resin beads into empty micro-fuge tube, desalt the supernatants by adding the equivalent of 100 microliters of an anion cation exchange resin beads and agitate.
Collect the samples by pipetting, and place them in new annotated tubes. Freeze dry the samples. Store dried samples at room temperature or 20 degree Celsius.
Mix the dried samples with 2 microliters of 1 molar sodium Cyanoborohydride and Tetrahydrofuran, and 2 microliters of 8 amino 1-3-6 pyrene trisulfonic acid. Incubate at 42 degrees Celsius for 16 hours in the dark. Add 46 microliters of ultra pure water to each sample.
To dilute the samples 50 times, add one microliter of the sample to 49 microliters of ultra pure water in 100 microliter microvials. In vortex to mix thoroughly. Place the samples in the dark for 5 to 10 minutes while setting the face.
To perform reverse polarity electrophoresis, set up the method, set the injection pressure to 0.5 pound force per square inch. Then set the polarity to reverse mode for separation. Dilute the N-linked carbohydrate separation buffer to one-third in ultra pure water.
Carry out the APTS labeled Glucans separation in the diluted buffer at 30 kilovolts in a bare fused silica capillary. Then set up the injection time, and start the analysis. Export the ASC and CDF files containing the electropherogram profile and integration data respectively.
Open the ASC file and draw the relative fluorescence unit according to the time chart. Open the CDF file. Proceed with a first automatic integration and adjust the following parameters with valley to valley integration and minimum area.
Check and correct any improper integration event manually. The fluorescence signals observed between 4.13 and 4.67 minutes originated from the unreacted APTS. The Aleutian time of labeled Maltohexaose DP 6 was estimated at 8.49 minutes.
The APTS labeled Glucans of bovine glycogen were identified based on the Aleutian time of DP 6. No trace of free Maltooligosaccharide was detected in the control sample in which glycogen was not incubated with debranching enzymes. The inset panel shows a separation of Glucan chains up to 44 DP.The chain length distribution is shown as the percentage of DP for each DP.The average chain length was calculated by summing each percentage chain times The corresponding degree of polymerization.
Face analysis showed that the most abundant Glucans are Maltose and rabbit liver, Maltohexaose in the oyster, and Maltoheptaose in the bovine liver. The chain length distributions of glycogen purified from wild type and mutant Cyanobacterial bacterial strains were determined using face analysis. Subtractive analyses were performed by subtracting the percent of each DP of wild type from the percent of each DP of mutants.
The average chain length distributions of glycogen from wild type and mutants of Synechocystis were normalized according to the maximum peak observed for each CLD. This technique allows a better understanding of human diseases associated with a defect in glycogen metabolism, particularly Andersen's disease disease, rich results from the accumulation of abnormal glycogen particles in cells.
