Before this, I used to detect hormones by using crab's hemolymph, but I am trying to detect hormone in crabs's larvae in my present study. So, I need the best method in handling larvae before proceed with the ELISA test. I have an inquiry about their preferable weight, either dry weight or wet weight? The solution to be added with the larvae? Can anyone please help me. Thank you very much.
Hi Amin,
What are you targeting with the ELISA? Hormones? Are you planning on homogenizing the larvae and doing an extraction? Depending on the hormone, you could just freeze your larvae and extract it at a later date. Then, when you're ready, you thaw your larvae, add the extraction buffer (PBS, methanol, etc) and continue your extraction.
Hi Danielle,
Thank you very much. I am targeting to know the levels of moulting hormone (20-Hydroxyecdysone) in the larvae. Yes, I am planning to homogenized and extract the larvae, but I still confuse with the extraction buffer. I also would like to know the weight of larvae needed to perform ELISA test. Yesterday, I found a journal by Planello et al. (2015) regarding Chironomus riparius larvae, they only used 1 larvae per sample. Can you suggest me any references to perform this ELISA test?
Hi Amin,
The attached insert from a ELISA kit from Cayman Chemical might be useful - there are many references. It might be worth doing some tests to see how much mass you need to start with. I work with fish homogenate and depending on the hormone I'm extracting, I use anywhere from 0.15 g to 0.5g of tissue. How much does 1 larvae weigh?
Since 20-Hydroxyecdysone is a steroid hormone, you could probably extract in methanol, blow it down using air or nitrogen, and then re-suspend in the kit's assay buffer. There will be some trial and error to determine input mass and final resuspension volume to achieve an appropriate concentration for your kit.
https://www.caymanchem.com/pdfs/501390.pdf
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