Phosphorylation-status of phospholamban and calsequestrin modifies their affinity towards commonly used antibodies.
Phospholamban (PLB) and calsequestrin (CSQ) play important roles in sarcoplasmic reticulum Ca(2+) transport and storage in cardiac muscle. Specific antibodies have been frequently used to quantitate CSQ and PLB protein levels. Here we demonstrate that two of the commonly available anti-PLB antibodies, anti-PLB-2D12 and anti-PLB-A1, show lower reactivity to phosphorylated than dephosphorylated PLB. A custom anti-PLB antibody, generated using a peptide corresponding to amino acids 2-14, is not affected by the phosphorylation state of PLB. In contrast, anti-CSQ reacts less with dephosphorylated CSQ than with phosphorylated CSQ. All three commercially available antibodies tested in this study have been widely used to quantify PLB and CSQ expression, and the results are integrated in many publications. Our studies reveal that the phosphorylation status of PLB and CSQ can affect antibody reactivity and may lead to over- or underestimation of the relative protein content and erroneous interpretation of data.
Huke
Sabine
S
Department of Physiology and Cell Biology, The Ohio State University College of Medicine and Public Health, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA.
Periasamy
Muthu
M
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002155:Calsequestrin / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism; D000099211:Phospholamban
D000906:Antibodies; D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D000099211:Phospholamban