Caspase-1 Assay Kit (Fluorometric) (ab39412)

The answers to your questions are as follows:


For doing the caspase-1 assay the cells are lysed and then treated with the substrate. Thus, cell permabilization is not an issue.
A reducing environment is required for the caspases to remain stable in solution and retain activity. This is why DTT is used.

The lysis buffer in the kit does not contain any protease inhibitors. The assay relies on keeping the samples on ice to minimize degradation, until the assay at 37C. You could add protease inhibitors to a portion of you lysates set aside for western blots immediately after homogenization, but do not add them to the portion you intend to use for the assay, as caspase activity could be inhibited.

For a western blot test, I suggest loading a lane in a gel with a sample that did not receive inhibitors, and compare the result to a lane that was loaded with sample + inhbitors, to see if protease inhibitors are necessary to keep caspase 2 intact.

ab39412 has sensitivity of 0.05 - 0.5 ng.

There is 1% Triton-x-100 in the lysis buffer. For best results we recommend using the cell lysis buffer provided. If absolutely necessary for the virus, you may use the RIPA buffer with this kit, but results may vary.

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Unfortunately we do not have a sensitivity range determined for this kit. Fold-increase in Caspase-1 activity can be determined by comparing the results of induced samples with the level of the untreated control. Please let me know if you have any more questions. 

Thank you for your inquiry. This result would depend on a number of factors including the cell type in which the Capase-1 activation is being studied, the levels of Caspase-1 activation after induction of apoptosis, time-course for Caspase-1 activation after apoptosis induction. We do have the following citations for this kit which will be of help to you to give you an idea: Caspase-1 Fluorometric Assay Kit Cheng, S. C. et. al. The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans. J. Leukoc. Biol., Aug 2011; 90: 357 - 366. Cihakova D et al (2008) Am. J. Pathol. 172: 1195-1208. Coletti, D., et al. (2002) EMBO J. 21:631-642. Duncan JA et al (2009) J. Immunol.; 182: 6460-6469Koenen, T. b. et. al. Hyperglycemia Activates Caspase-1 and TXNIP-Mediated IL-1β Transcription in Human Adipose Tissue. Diabetes, Feb 2011; 60: 517 - 524.  I hope this information helps. Please contact us with any other questions.

Thank you for your call today. I have heard back from the lab about your questions: 1) For tissues, homogenize 10 mg of tissue in 200 ul of lysis buffer. Spin out debris and add 50 ul of supernatant into a well of the 96-well plate then proceed from Step 4 on the protocol. 2) You can use the lysate for Western blot. I hope this information is helpful, but please let me know if you have any further questions.

Thank you for your enquiry. The colorimetric assay has sensitivity of about 5 ng and fluorometric is about 10-100 fold more sensitive. Fluorometric assay is recommended for tissue samples. I hope this information helps. Please do not hesitate to contact us if you need any more advice or information.