Development and Characterization of Monoclonal Antibodies to Detect Klotho

Abstract

Although antibodies are commercially available to allow investigation into the biology of the age-regulating protein Klotho, problems with antibody specificity and application functionality are significant barriers to progress. Chief among these limitations is the inability of current tools to allow in vivo validation of binding partners originally identified through transfection of tagged proteins. To overcome this barrier, we generated a series of hybridoma cell lines by immunizing rats with a GST-KL1 fusion protein. Purified antibodies generated from these cell lines differentially detect human or mouse Klotho protein via Western blot, immunocyto/histochemistry, and immunoprecipitation. Specificity of antibody binding to Klotho was confirmed by mass spectrometry following immunoprecipitation. With this confidence in antibody specificity, co-immunoprecipitation was utilized to validate the interaction of Klotho/FGFR and Klotho/wnt7a in mouse kidney lysates.

Klotho regulates CA1 hippocampal synaptic plasticity

“…Consistent with previous reports (Li et al, 2004, Maltare et al, 2014), choroid plexus KL immunoreactivity is most intense (Figure 1A). Although less highly expressed than choroid plexus, throughout subfields of the hippocampus, neuronal cell bodies and processes express KL (Figure 1B).…”

“…Membranes were blocked with 5% non-fat dry milk in Tris buffered saline with 0.1% Tween 20 (TBST). Membranes were incubated in primary antibody (in 0.3% BSA in TBST) at 4°C overnight using antibodies to detect KL (KL2-34 (Maltare et al, 2014)), β-tubulin (Developmental Studies Hybridoma Bank, E7, Iowa City, IA), synaptophysin (Abcam, AB52636, Cambridge, MA), GluN1 (Neuromab, 75–272, Davis, CA) and SNAP25 (SC73044, Santa Cruz). Secondary antibodies conjugated to HRP were detected by chemiluminescence (Immobilon, Millipore, Billerica, MA) and exposure to film.…”

FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model

“…The precipitate was washed 3 times with 1% Triton X-100 in PBS (pH 7.4) and once with PBS (pH 7.4), mixed with SDS-PAGE sample buffer, and subjected to 8% SDS-PAGE. After transferring onto a nitrocellulose membrane, Klotho was detected using anti-Klotho rat mAb (KM2076, TransGenic Inc.) (85) or anti-Klotho KL1 rat mAb (KL-234, provided by Gwendalyn D. King at University of Alabama at Birmingham, Birmingham, Alabama, USA) (86), and the density of immunoreactive Klotho bands was analyzed by ImageJ software (NIH).…”

Klotho regulates postnatal neurogenesis and protects against age-related spatial memory loss

“…ND1 was detected using diaminobenzidine (DAB)(King et al 2008; King et al 2011). GFP and GFAP co-localization with KL was detected using 10μm thick paraffin sections and anti-KL antibody (R and D Systems, Minneapolis, MN AF1819) as reported(Maltare et al 2014). This KL antibody is directed against the amino terminus of the KL protein and thus detects all protein forms.…”