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Nitric oxide (NO) is produced by three distinct isoforms of nitric oxide synthase and functions as a key signaling molecule in physiology and pathophysiology.1,2 NO transduces its effects by reacting either directly with heme and non-
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1. Alderton, W.K., Cooper, C.E., and Knowles, R.G. Nitric oxide synthases: Structure, function, and inhibition Biochemistry Journal 357, 593-615 (2001).
2. Bredt, D.S. Endogenous nitric oxide synthesis: Biological functions and pathophysiology Free Radical Research 31, 577-596 (1999).
Ckless, K., Reynaert, N.L., Taatjes, D.J., et al. In situ detection and visualization of S-
Jaffrey, S.R., Erdjument-
In the S-Nitrosylated Protein Detection Kit is there any point early in the procedure where the protocol can be stopped overnight?
It is possible to stop the procedure at either of the two acetone protein precipitations (step 7 or step 13 under the Sample Preparation section). At either point you can leave the precipitates at -20°C overnight.
If no dark room is available, how important is the performance of steps 1-12 under indirect light in the S-Nitrosylated Protein Detection Kit protocol?
If you can turn off some of the overhead lights in the lab and avoid using a lamp directly on the bench, that will help.
In the S-Nitrosylated Protein Detection Kit there is not a protocol for use of a whole tissue homogenate. There is a protocol for isolated tissue cell pellet, but nothing starting from whole tissues. Is there a way to use this kit?
For a soft tissue where cells can be isolated one may: isolate, wash and pellet the cells, pick up at step 3 to lyse cells and block free thiols. For solid tissue samples, homogenize at 0.1 g/ml buffer in 20 mM Tris pH 8.0, or P-SNO Cell Lysis Buffer (Buffer A). Centrifuge here to eliminate major debris (1000xg, 10 minutes). Repeat the homogenization with the ice-cold supernatant and sonicate if needed (to ensure complete lysis, check for remaining intact cells by microscopy). Quantify the protein concentration and dilute to 2 mg/ml if needed. Treat the sample going forward as a cell lysate by adding the reconstituted blocking reagent (1 mL stock solution) to the current sample volume in 1:9 proportion.
For the S-Nitrosylated Protein Detection Assay Kit is it is okay to have β-mercaptoethanol in the Laemmli buffer?
Yes, it is okay to have β-mercaptoethanol in the Laemmli buffer in the S-Nitrosylated Protein Detection Assay Kit. The Biotin will not dissociate from the protein.
What sample types can be used with the S-Nitrosylation Protein Detection Kit?
The protocol in the S-Nitrosylated Protein Detection Kit (cat. 10006518) refers to use of a cell pellet which can be obtained from cultured cells, or blood cells. Alternatively, the assay can be used with purified protein. If using a purified protein, begin by resuspending the protein sample and incubating at 2mg/mL in Buffer A containing Blocking Reagent; this will keep the blocking reagent in excess. At this point, free thiols on the protein are blocked and you can continue with the acetone precipitations in step 6. For gel analysis, load only 1-5 µg of protein per lane to avoid overloading the protein.