Which method is the best to separate LDH isoenymes in med lab?
Ali,
We have used predominantly electrophoretic method for the separation of LDH isoenzymes, which appeared not so hard even in med lab environment. Due to their different amino acid compositions the LDH isoenzymes can be separated in polyacrylamide used as a matrix. In an electric field at pH 8.8 the LDH1 migrates at the fastest rate towards the anode, while the LDH5 is the slowest isoenzyme. After electrophoresis the LDH isoenzymes can be visualized by an activity staining process where the product of the enzymatic reaction is a water-insoluble dye precipitating in the gel where the LDH enzymes are located.
We used to use the following materials: 1. pre-cast 7.5% polyacrylamide gel slab; 2. tank buffer (2.4 g Tris base, 11.6 g glycine/liter); 3. samples made from liver, heart muscle, and kidney in sample loading buffer; 4. 1 M Tris- HCl, pH 8.0; 5. NAD, 10 mM; 6. tetrazolium-blue, 1 mg/ml; 7. phenazine-methosulphate, 1.6 mg/ml; 8. 1 M Na-lactate.
The sample wells are rinsed out with the tank buffer and the gels are placed into the electrophoretic unit. Load 5 l of each sample into different wells of the gel. Pour tank buffer into the reservoirs and connect the electric cables. The positive pole is at the bottom. Turn the power source on and set the current at 12 mA. Run the electrophoresis for 90 minutes. Turn off the power, separate the two plastic plates by prying them apart with a spatula and place the gel into the developing chamber which already contains the developer solution (H2O 18.4 ml, 1 M Tris 4 ml, tetrazolium-blue 12 ml, phenazinemethosulphate 4 ml, Na-lactate 4 ml and NAD 1.3 ml). Incubate at 40 C to develop color reaction for 20 minutes. In the color reaction NAD and lactate serve as substrates, phenazine-methosulphate is the primary electron acceptor and tetrazolium-blue is the final electron acceptor. Wash the gel with water.
Best wishes,
Ilya
Hi there,
You might use commercial kit for LDH isoenzyme resolution:
http://www.interlab-srl.com/en/test-menu/ldh-isoenzymes-electrophoresis-kit
Dear llya and Dominique
Thank you for your greate explanations. Ali
Dear llya and Dominique
Thank you for your greate explanations. Ali
6/25/15
Dear Dr. Moshtaghie,
The replies by Drs. Liger and Tsyrlov are both excellent suggestions. If the isoenzymes can be resolved by electrophoresis, their pI's must be different enough that you should be able to also resolve AND isolate them via anion exchange chromatography. An anion exchanger such as DEAE (or similar resins) used with gradient elution might work for you. If you have access to an automated FPLC (Fast Protein Liquid Chromatography) system (e.g., AKTA), this would be helpful for you. The system provides automation for loading the chromatography column, elution with buffer, switching gradients, etc. and pumps eluents to a fraction collector. It also is equipped with "real time" spectrophotometric monitoring (e.g., A280) enabling you to seed individual peaks as they elute. This would allow you to trouble-shoot different gradients to maximize resolution of the isoenzymes.
I hope this will help you.
Bill Colonna, Iowa State University, Ames, IA, USA
You may use the Sebia kit for Hydrasys. It is semi-automatised.
www.sebia-usa.com/products/ISO-LDH.html
For more manual, use Helena Laboratories
www.helena.com/Procedures/Pro077Rev3.pd
Dear Ilya,
I attach this paper which is in the spirit of Bill Colonna here above. Ion exchange chromatography is the way to go.
Best regards,
Jon
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Dear colleagues
Thanks from all of you for giving very good ideas
@Ali Asghar Moshtaghie : Which technique did you eventually employ to separate LDH isoenzymes?
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