Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.
Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.
1 Department of Chemistry and Biochemistry, Texas State University , San Marcos, Texas.
Michael A
MA
Villarreal
1 Department of Chemistry and Biochemistry, Texas State University , San Marcos, Texas.
Nicole M
NM
Biediger
1 Department of Chemistry and Biochemistry, Texas State University , San Marcos, Texas.
Natalie A
NA
Bonner
1 Department of Chemistry and Biochemistry, Texas State University , San Marcos, Texas.
Jennifer N
JN
Miller
1 Department of Chemistry and Biochemistry, Texas State University , San Marcos, Texas.
Samantha K
SK
Zepeda
1 Department of Chemistry and Biochemistry, Texas State University , San Marcos, Texas.|2 Department of Biology, Texas State University , San Marcos, Texas.
Benjamin J
BJ
Ricard
2 Department of Biology, Texas State University , San Marcos, Texas.
Dana M
DM
García
1 Department of Chemistry and Biochemistry, Texas State University , San Marcos, Texas.
Karen A
KA
Lewis
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D018604:Epitope Mapping / Q000379:methods*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D044122:Receptor, Melatonin, MT1 / Q000276:immunology; D044123:Receptor, Melatonin, MT2 / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017385:Sequence Homology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000276:immunology*; D029961:Zebrafish Proteins / Q000276:immunology*
D000906:Antibodies; C000610981:MTNR1A protein, human; C573871:MTNR1B protein, human; D044122:Receptor, Melatonin, MT1; D044123:Receptor, Melatonin, MT2; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D029961:Zebrafish Proteins; D006639:Histidine
antibody specificity; antigen specificity; epitope mapping; immunohistochemistry; recombinant protein