Calpain Activity Assay Kit (ab65308)

Ab65308 measures overall calpain activity. It will measure activity from any active Calpain isoform in the sample. Calpain I and II are mainly expressed in the central nervous system. So depending on what your sample is, the calpain isoform can differ. Also, Calpain I and II (also called mu-calpain and m-calpain respectively) are activated by micro- and nearly millimolar concentrations of Ca2+ within the cell. To determine Calpain I activity exclusively either you have to make sure that the calcium concentration is in micromolar range in the sample or you have to purify calpain I specifically to test its activity.

There is ˜50 mM Calcium in the 10X reaction buffer but not in Extraction buffer. The Extraction buffer however is supplied with optimal concentrations of EDTA and EGTA to chelate the calcium to avoid any autoactivation of calpain during the extraction procedure.

We do not provide separate source of calcium other than what is present in buffer.

For the Calpain assay we highly recommend using fresh tissues or tissue samples snap-frozen after collection. The frozen tissue can be ground and then homogenized in the extraction buffer.

One point to note is : Inactive calpain remains associated to the cell membrane and active calpain is released into the cytosol. Using an extraction buffer which pokes holes in the cell membrane only the active calpain in the cytosol will leak out and get assayed. The Extraction Buffer provided with the kit specifically extracts cytosolic proteins without contaminations of cell membrane and lysosome proteases. The Extraction Buffer also prevents auto-activation of calpain during the extraction procedure. Thus, the kit detects only activated calpain in cytosol upon treatment of cells with inducers (e.g., chemicals or drugs). Any cell lysis buffer will not distinguish between inactive and active calpain. When using a tissue homogenate there might be some autoactivation of calpain.

The success of this kit with frozen samples depends on how well the samples were frozen, for how long they had been frozen, if they underwent repeated freeze thaw and so on.

Immediate freezing of the sample in aliquots and storing for up to 2 months without any freeze thaw is ideal.

The lab also confirmed that more than 1-2% DMSO will interfere with the assay.

This kit has the substrate bound to the AFC fluorophore.

The success of this kit with frozen samples depends on how well the samples were frozen, for how long they had been frozen, if they underwent repeated freeze thaw and so on.
Immediate freezing of the sample in aliquots and storing for up to 2 months without any freeze thaw is ideal.

I can confirm that for the sample preparation the cells will not need to be trypsinized. They can be scraped from the flask after after adding the extraction buffer and incubated, but keep in mind that this would dilute the sample for the activity assay. It would require a few mls of the extraction buffer to cover the cells. An alternative is to wash the cells with PBS. Then scrape the cells in PBS, spin it down, remove PBS and resuspend cells in extraction buffer.

This Calpain Activity Assay Kit includes Active Calpain I as positive control.

Thank you once again for your enquiries.

I have obtained some further information for you.

Unfortunately, I cannot disclose the contents of the extraction buffer due to proprietary restrictions, but I can assure you that it is strong enough to lyse cells. Nothing needs to be added to it.

The large pellet from the resting platelets could possibly be a result of improper lysis. Was the pellet observation reproducible? If you are not sure of cell lysis just after the 20 min incubation on ice, I can recommend to use a homogenizer or a fine needle syringe to obtain full lysis of the sample. This kit has been used exactly as recommended, without any modifications multiple times. I have included some references below which have used this kit. I hope these would be helpful:



- Babbin BA et al (2009) Am. J. Pathol.; 174: 436 - 448.

- De la Fuente MA et al. (2007) PNAS 104: 926-931.

- Glading, A., et al. (2004) Mol. Cell. Biol. 24: 2499-2512.

- Guha, P. et al. Calpain and Caspase Orchestrated Death Signal to Accomplish Apoptosis Induced by Resveratrol and Its Novel Analog Hydroxstilbene-1 in Cancer Cells. J. Pharmacol. Exp. Ther., 2010; 334: 381-394.

- Huang, W. et al.The Interferon Consensus Sequence Binding Protein (ICSBP) Decreases β -Catenin-Activity in Myeloid Cells by Repressing GAS2 Transcription. Mol. Cell. Biol., 2010; 10.1128/MCB.01595-09.

- Lee W-K et al. (2006) Am. J. Physiol. Renal Physiol. 291: F823-F832.

- Leloup, L. et al. m-Calpain Activation Is Regulated by Its Membrane Localization and by Its Binding to Phasphatidylinositol 4,5-Bisphosphate. J. Biol. Chem., 2010; 285: 33549-33566.

- Liou, A. K. F., et al. (2005) FASEB J. 10.1096/fj.04-3258fje.

- Liu, J. et al., Tiam1-regulated osteopontin in senescent fibroblasts contributes to the migration and invasion of associated epithelial cells, J. Cell Sci., Jan 2012; 125: 376 - 386.

- Lee W-K et al. (2007) Am. J. Physiol. Cell Physiol. 293: C839-C847.

- Mani SK et al (2008) Am J Physiol Heart Cir Physiol 295: H314-H326.

- Pareek T.K. et al. (2006) PNAS 103: 791-796.

- Peyrou M et al. (2007) Toxicol. Sci. 99: 346 353.

- Singh RB et al (2008) J Appl Physiol; 105: 1779 - 1787.

- Song, Y., et al. (2005) J. Clin. Invest. 115:451-458.

- Tsai, J. Y. et. al. EGb761 ameliorates the formation of foam cells by regulating the expression of SR-A and ABCA1: role of haem oxygenase-1. Cardiovasc Res, Dec 2010; 88: 415 - 423.

- Tsai, J. et al. EGb761 Ameliorates the Formation of Foam Cells by Regulating the Expression of SR-A and ABCA1: Role of Haem Oxygenase-1. Cardiovasc. Res., 2010; 10.1093/cvr/cvq226.

- Undyala VV et al (2008) J. Cell Sci.; 121: 3581 - 3588.

- Watterson, T. et al. Urban Particulate Matter Causes ER Stress and the Unfolded Protein Response in Human Lung Cells. Toxicol. Sci. 2009; 112: 111-122.

- Whiteman, M., et al. (2004) FASEB J. 10.1096/fj.03-1096fje.

- Yeh, Y. H. et. al. Transforming growth factor-β and oxidative stress mediate tachycardia-induced cellular remodelling in cultured atrial-derived myocytes. Cardiovasc Res, Feb 2011; 0.1093/cvr/cvr041.

- Yeh, Y. H. et. al. Transforming growth factor-β and oxidative stress mediate tachycardia-induced cellular remodelling in cultured atrial-derived myocytes. Cardiovasc Res, Jul 2011; 91: 62 - 70.

Hope this information has been useful for you. Please do not hesitate to let me know if you have any other questions or if you continue to have difficulties with the samples or the kit.