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First what is the substrate? I ask because often IEPD or IETD are used as substrates for Granzyme B, but these are also cleaved by caspases. Do you have a more complete protocol? Specifically for sample preparation what lysis buffers/conditions do you recommend?
Asked on Apr 25 2013
Due to proprietary restrictions I cannot disclose what exactly the substrate is in this kit, but I can assure you that it is granzyme B specific.
Here is the protocol for processing cells for this assay:
For cell samples: Start with ˜2X106 cells, suspend the cell pellet 500 μl (or ˜4 volumes) of the assay buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Use the supernatant for your subsequent assays. Appropriate dilutions of the sample must be tested in order to ensure the readings will fall within the linear range of the standard curve.
Answered on Apr 25 2013