Hey everyone!

I need some advice for the determination of the mutant adult zebrafish I generated (besides DNA Sequencing).

I want to use Western blot to check if the protein is really gone. I already tried to do the lysis of one fish, but the blot in the end looked very blurry and faint compared to one I did with embryo lysate where I could see clear bands.

The protocol I used was this: Lysis of the (frozen) adult fish with 700 µl NP-40 buffer (including protease inhibitor) using a Tissue lyser II and a metal bead for 2 times, 2 min, 25 Hz. Afterwards everything was centrifuged for 5min, 4°C and 14000 rpm and supernatant sotred at -80°C. Protein conentration was determined using BCA protein assay Kit.

Maybe the lysis was not sufficient enough due to the size of the fish (up to 2 cm/60 mg) or I carried too many interfering substances within the supernatant.

For the RNA Isolation i want to do the lysis the same way (so with a metal bead, frozen fish), but using Qiazol lysis reagent and the protocol from the miRNeasy Kit (Qiagen).

Thank you very much for any suggestions, help and critiques!