Dear colleagues, does anyone know a reliable method for the extraction of melanin (from both cuticle and formed by immune activated phenoloxidase in the body cavity) from whole insects?
Thank you very much
Dear Christoph-Rüdiger von Bredow
For determining total phenoloxidase you should freeze your samples (at nitrogen tank or -80 Celsius). When you measuring Phenoloxidase, firstly homogenised your samples; after centrifuge at 10 000 rpm (+4 Celsius) for 15 min. Take supernatant and placed ice cold micro centrifuge tube. That is pure samples; if you like can dilute with phosphate buffer. If you use ELISA plate reader 40 microliter samples placed in 160 microliter L-DOPA in culture plate and read at 492 nm. If use spectrophotometer calculate your solutions. You may also use given article.
"Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs"
Dear Serhat,
thank you very much for the PO-activity protocol. I probably did not make my question clear enough, since I wish to know how already polymerized melanin, which is present in sclerotised cuticle as well as a result of the PO-mediated tyrosine oxidation, can be isolated. But, again, I wish to thank you since your protocol may come in handy for other assays like determination of PO-activity in response to immune challenge.
Dear Christoph-Rüdiger von Bredow
Thank you for you explanation, I have not any protocol for your question. I'm working on immune response cellular and humoral. I hope some one can answer your question, fallowing with interest.
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