Dear all,
We are trying to quantify different proteins in 6 dpf zebrafish larvae. After exposure we collect the larvae in PBS which is then replaced by RIPA buffer containing PMSF and a cocktail of inhibitors. Following homogeneization and centrifugation, we quantify protein by BCA method and apply 50 ug in a 4/12% SDS gel. After running the gel and performing the transfer, we proceed with the common blocking (5% milk), primary antibody incubation (in 5% BSA) overnight at 4 ºC, secondary antibody incubation (in 5% milk) and proceed with the detection using a chromogenic substrate. Yet, and after deeply optimize every step in the western blot procedure, we are still not getting protein signals except for the GAPDH antibody.
We have tried this same procedure with adult zebrafish brain and we get bands for some of the antibodies that we're testing but the larvae do not show any band (an example can be found attached).
Does anybody have a possible explanation for not getting any signal from larval samples? Is this possibly related to antibodies specificity for each organ?
Thanks in advance
Did you de-yolk the larvae? If not, the larval lanes contain mostly yolk protein. So you will lose signal for your target protein because the concentration is too low. See Fig. 3 this paper:
Article Proteomics of early zebrafish embryos
Dear Elisabeth E LeClair,
Thank you for your reply. However, the mentioned article and the mentioned procedure is valid for early developmental stages in which the yolk may be a problem for proteomic analysis. Yet, I'm using 6 dpf larvae which we do not de-yolk as there is no information that the yolk could interfere in the analysis.
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