I am using the CRISPR/Cas system and will have zebrafish embryos with introduced indels in my gene of interest. I have a really good antibody for my gene so I would like to western blot some embryos to see if the indel knocks out the gene. Of course I can (and will sequence) some embryos to look for frameshift but since perhaps the fish could compensate with alternative splicing or something I would like to western blot as well. Since any non-indel containing embryos would show a band I can't combine embryos and I won't be sure which ones contain indels since I can't do genetics and protein on the same embyro (I am assuming not enough material). What is the best way to get the maximum amount of protein from a single embryo (5 dpf) to run on a gel for western?
Homogenize your embryo in lysis buffer such as RIPA buffer and then proceed to protein isolation, estimation and sample preparation for western blotting the way you would for a cell line.
I am not much aware of the fish genetics but can't you genotype your embryos and then separate the knockouts and wild type embryos and then perform westerns with both embryos to compare the protein expression of your gene of interest.
Thank you for your answer. The problem with trying to genotype them first, is that usually I use the whole embryo to extract the DNA so then I would not be able to get protein from the same sample.
PCR can work on single cells, so you do not need much for genotyping. They do sell kits that give you DNA and protein from the same sample, but here's a cheaper option
- Cut off each larval tail and isolate DNA. Homogenize the rest of the embryo as for Western and freeze the homogenate.
- Once you know the individual genotypes, you can pool similar ones to load in your protein lanes. 5-10 embryos per lane should be sufficient. If your antibody is good and you use sensitive detection method, perhaps less.
Good luck with your experiment.
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