Lactate Dehydrogenase ProtocolLactate dehydrogenase is an enzyme which is useful for monitoring cell death and disruption. Kits are available to run LDH assays from many biological suppliers. Alternatively, the assay can be developed in-house more cost effectively. The following protocol is for an in-house recipe. LDH assays are used routinely to assess the efficiency of homogenization methods in our product development lab. For more information on OPS Diagnostics' homogenization products, click here.
|200 mM TRIS, pH 8||Most LDH works well at this pH. To prepare, add 22.2 gm of Tris-HCl (Sigma-Aldrich, T-3253 or equivalent) and 10.6 gm of Tris-base (Sigma-Aldrich, T4661 or equivalent) to 1 L water.|
|50 mM Li Lactate||Prepare by adding 49 mg lithium lactate (Sigma L-1500 or equivalent) into 2.5 ml of water.|
The following assay is for a microplate reader, though it can be scaled up for cuvettes or tubes. A good wavelength for measuring OD is 490 nm. Wells should be prepared for all samples, ran in triplicate, plus three negative controls using water. If LDH is available, three positive controls can be ran as well.
1. Add all the reagents first, followed by the cell lysates. Do not cross contaminate the wells.
|200 mM TRIS, pH 8||50 µl|
|50 mM Lithium Lactate||50 µl|
|PMS, INT, NAD||50 µl|
2. Add 50 µl sample or control. The assay can be run as an end point assay (wait 5 minutes and read the plate) or kinetic assay based on the microplate reader. Most accurate measurements determine the maximum slope during a kinetic assay.
An example of using the LDH assay for measuring homogenization efficiency can be found at the following link, Homogenization of Mouse Muscle: A Comparison of Methods.