Hi Nuria,

I have experienced this problem before. Within larval extract you will have an amount of haemolymph, which contains circulatory immune cells known as hemocytes. A particular sub population of hemocytes, called crystal cells, contain crystals of pro-phenol oxidase that once ruptured will release the pro-phenol oxidase to induce the melanisation cascade. This produces melanin, which is the black substance you are observing. Larval haemolymph is also known to undergo 'auto-melanisation', which is a global response from damaged tissues and circulatory hemocytes together, but appears to be larval specific. Dare I say, your freeze-thaw procedure is adding to this affect by rupturing more crystal cells.

The only things that I can suggest would be to conduct the extract with pH balanced PBS or HBSS+HEPES (pH 7.4) as well as on ice during the preparation phase. And then could you not continue through with your reaction? Speed would be the key, usually whole sample melanisation would occur at ~4-5 hours at room temperature. Otherwise the use of Urea will inhibit the melanisation response. When I used to do this form of work I would use poly-thiol urea (PTU) at very low concentrations, which would always vary dependent on the amount of haemolymph extracted. I would suggest trial and error with the urea, plus controls in the forms of urea only, and -urea sample. Im not sure what reactions you are performing so the urea may not be the best idea, but hopefully it will work out for you!

Best of luck,

Chris

Hi Chris,

Thanks for your answer. I guess this means my samples are not usable any more. I'll try your suggestions next time.

Best

Nuria

Dear Nurea,

I think the suggestion to use phenyl-thio-urea is the best choice, if you add a trace before centrifugation to deactivate the phenoloxidae activity in the extract. Another advice is to run every thing in cold condition, meaning crushing of larvae in ice then ice-cold centrifugation and aliquot your samples for the experiments, so one sample comes from freezer to immediate use.

best wishes

Wesam

Hey Wesam!  did you checked for phenyl-thio-urea personally or do you have any reference paper that had shown in there protocol. 

Hello everyone,

can you give me a concentration of poly-thiol urea to use in protein sample extractions from Drosophil larvae ? Due to melaniation, I can not measure the protein concentration in my samples. It is, thus, impossible to run a correct western blot.

Thank you.

I too got the same problem during preparation of extract

0.01% of Phenylthiourea worked for me. It inhibited melanization for at least 24hrs. I stopped the experiment after 24hrs. The experiment was done by homogenizing 10 Drosophila L3 larvae. Optimize accordingly.