title: Comparison of Equivalence between Two Commercially Available S499-Phosphorylated FMRP Antibodies in Mice.
abstract: Fragile X syndrome (FXS) develops from excessive trinucleotide CGG repeats in the 5'-untranslated region at Xq27.3 of the Fmr-1 gene, which functionally silences its expression and prevents transcription of its protein. This disorder is the most prominent form of heritable intellectual deficiency, affecting roughly 1 in 5,000 males and 1 in 10,000 females globally. Antibody specificity and selectivity are essential for investigating changes in intracellular protein signaling and phosphorylation status of the Fragile X Mental Retardation Protein (FMRP). Currently, both PhosphoSolutions® and abcam® produce commercially available S499-phosphorylated FMRP specific antibodies. The antibody from PhosphoSolutions® has been validated in previous studies; however, the antibody from abcam® antibody has yet to receive similar validation. This study aims to determine whether these two antibodies are true equivalents through western blot analysis of both NS-Pten knockout (KO) and Fmr-1 KO mice strains. We prepared hippocampal synaptosomal preparations and probed the samples using total FMRP, abcam® phosphorylated FMRP, and PhosphoSolutions® phosphorylated FMRP antibodies. We found that there was a significant increase in phosphorylated FMRP levels using the abcam® and PhosphoSolutions® antibodies in the NS-Pten KO mice compared to wildtype mice. However, there was much more variability using the abcam® antibody. Furthermore, there was a band present in the Fmr-1 KO for the phosphorylated FMRP site using the abcam® antibody for western blotting but not for the PhosphoSolutions® antibody. Our findings strongly suggest that the antibody from abcam® is neither specific nor selective for its advertised targeted substrate, S499-phosphorylated FMRP.
authors.affiliation: Department of Psychology and Neuroscience, Baylor University, Waco, Texas, United States of America.
authors.forename: Conner D
authors.initials: CD
authors.lastname: Reynolds
authors.affiliation: Institute of Biomedical Sciences, Baylor University, Waco, Texas, United States of America.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Smith
authors.affiliation: Department of Psychology and Neuroscience, Baylor University, Waco, Texas, United States of America.
authors.forename: Taylor S
authors.initials: TS
authors.lastname: Jefferson
authors.affiliation: Department of Psychology and Neuroscience, Baylor University, Waco, Texas, United States of America.|Institute of Biomedical Sciences, Baylor University, Waco, Texas, United States of America.
authors.forename: Joaquin N
authors.initials: JN
authors.lastname: Lugo
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000918:Antibody Specificity; D015153:Blotting, Western / Q000592:standards*; D004195:Disease Models, Animal; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D005600:Fragile X Syndrome / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D017353:Gene Deletion; D015870:Gene Expression; D006624:Hippocampus / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D051059:PTEN Phosphohydrolase / Q000172:deficiency / Q000235:genetics; D010766:Phosphorylation; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D013810:Therapeutic Equivalency
chemical_list: D000906:Antibodies; D051860:Fragile X Messenger Ribonucleoprotein 1; D051059:PTEN Phosphohydrolase; C497684:Fmr1 protein, mouse; C487757:Pten protein, mouse