Antibody detection of Col11a1a protein by immunoblot and confirmed by mass spectrometry. A The antibody recognized protein bands migrating with apparent molecular weights of 100 and 35 kDa from 24 hpf zebrafish total lysate. Proteins extracted from the ECM contained the 100 kDa that was converted to a 35 kDa band upon treatment with collagenase. In the presence of a large excess of the peptide used as the antigen, the 100 kDa and 35 kDa bands from total lysate are not visible on the immunoblot, indicating competition by the peptide for the antigen binding site on the antibody. See Addiitonal file 1: Figure S1 for full length original unprocessed blots. B Representative immunoblot of proteins extracted from wildtype, heterozygous, and homozygous knockout zebrafish. The 100 kDa fragment of Col11a1a is apparent in lysate from 72 hpf zebrafish with quantitatively minor bands at the position of full-length Col11a1a. C Structural model of the amino propeptide domain of Col11a1a. Peptides detected by mass spectrometry are indicated in their respective locations of the exons encoding the protein. D Protein sequence of Col11a1a amino propeptide domain with grey shading indicates the sequence coverage used to confirm the identity of the protein recognized by the new antibody as Col11a1a. E Quantification of immunoblot band intensity from proteins extracted from wildtype (WT), heterozygotes, and homozygous knockout embryos. The NTD fragment and full-length molecule were quantified from three individual blots by densitometry, calculating average and standard deviation. Absence of these protein bands from homozygous knockout embryos confirmed specificity of the new antibody. WT wildtype, HET heterozygous, KO knockout, MW molecular weight markers
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