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<strong> Figure 1 WB Validation in Human Kidney </strong><br>Loading: 15 μg of lysate Antibodies: Nephrin, 2265, 2 μ g/mL , 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 WB Validation in Mouse Kidney </strong><br>Loading: 15 μg of lysate Antibodies: Nephrin, 2265, 2 μg/mL , 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 WB Validation in Rat Brain </strong><br>Loading: 15 μg of lysate Antibodies: Nephrin, 2265, 2 μg/mL , 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 6 Immunohistochemistry Validation of Nephrin in Human Kidney</strong><br> Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-Nephrin antibody (2265) at 1 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 6 Immunohistochemistry Validation of Nephrin in Mouse Kidney</strong><br> Immunohistochemical analysis of paraffin-embedded mouse kidney tissue using anti-Nephrin antibody (2265) at 1 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 6 Immunohistochemistry Validation of Nephrin in Rat Kidney</strong><br> Immunohistochemical analysis of paraffin-embedded rat kidney tissue using anti-Nephrin antibody (2265) at 1 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 7 Apoptosis Assay Validation of Nephrin in Mouse Glomerulus (Chen et al., 2017)</strong><br>Glomerular cells of Atgl (-/-) mice were double labeled with TUNEL staining (dark brown nucleus indicated by red arrows) and immunofluorescence staining of nephrin detected by anti-nephrin antibodies (2265) (pink cytoplasm indicated by green arrows) as a marker for podocytes. Colocalization of TUNEL-positive cells and nephrin proved that apoptosis was induced in Atgl(-/-) mice as compared to WT mice.
<strong>Figure 8 Immunolocalization Validation of Nephrin in Human Placenta (Yun et al., 2015)</strong><br>Immunofluorescence staining showed Nephrin expression detected by anti-nephrin antibodies (2265) was clearly localized in villi (A-C) and fetal membranes, Amnion (J-L) and Chorion (M-O). The staining was markedly positive at apical membrane of villi (arrows in B and C) and amnion (arrow in L), and in the stromal cells of chorion (small arrow in O).
<strong>Figure 9 Immunofluorescence Validation of Nephrin in Mouse Podocyte (Li et al, 2013) </strong><br> Double immunofluorescence analysis of podocytic membrane protein nephrin (red) and nuclei stained with DAPI (blue). The presence of high glucose (HG) and neutralizing antibody (NtAb) which blocked epithelial growth factor(EGF) decreased nephrin expression while mesenchymal stem cells-conditioned medium (MSCs-CM) and recombinant human EGF (rhEGF) prevented the effect.
<strong>Figure 10 Induced Expression of Nephrin by Curcumin Treatment in the Renal Tissues of Type 1 Diabetic Rats (Soetikno et al., 2013) </strong><br>Nephrin expression detected by anti-nephrin antibodies in type 1 diabetic rats. Nephrin was down-regulated in the vehicle-treated diabetic rats as compared to the control nondiabetic rats. However, this decreasein nephrin protein expression was markedly increased by curcumin treatment (P<.05) to near-normal levels. (n=5 in each group)
<strong>Figure 11 Immunofluorescence and Localization Validation of Nephrin in Cultured Rat Podocytes (Piwkowska et al., 2012)</strong><br>Immunofluorescence staining showed Nephrin expression (green) detected by anti-nephrin antibodies and PKGIalpha (red). The co-localization of two antibodies (yellow) in rat podocytes was observed particularly at the tips of the cell processes.
<strong>Figure 12 WB Validation of Nephrin in Glomeruli of Zucker Obese (ZO) and Zucker Lean (ZL) Rats (Piwkowska et al., 2013)</strong><br>The expression of nephrin detected by anti-nephrin antibodies did not change in ZO rats as compared to the control rats.
<strong>Figure 13 Immunohistochemistry Validation of Nephrin in Mouse Kidney (Toyama et al., 2012) </strong><br>Protein analysis for nephrin by immunohistochemistry with anti-nephrin antibodies in the kidney of wild-type or AMPD2-deficient mice at 2, 12 or 24 weeks of age. No difference between wild-type andAMPD2-deficient mice at any age was observed.
<strong>Figure 14 Regulated Expression Validation of Nephrin in Mouse Podocyte Cells Cultured in Normal Glucose (NG) Medium or High Glucose (HG) Medium (Huang et al., 2019) </strong><br>Western Blot analysis was used to access the protein expression level of nephrin with anti-nephrin antibodies. Nephrin expression was down-regulated by PEGF treatment (NGP or HGP), which was reversed by the addition of C3 transferase.
Figure 1 WB Validation in Human Kidney
Loading: 15 μg of lysate Antibodies: Nephrin, 2265, 2 μ g/mL , 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat Anti-Rabbit IgG HRP conjugate at 1:10000 dilution.
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Nephrin Antibody

CATALOG NUMBER: 2265

Clonality:
Polyclonal
Tested Applications:
IHC-P, WB
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Conjugate:
Unconjugated
Specifications
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Immunogen:
Anti-Nephrin antibody (2265) was raised against a peptide corresponding to 14 amino acids near the carboxy terminus of human Nephrin.
The immunogen is located within the last 50 amino acids of Nephrin.
Conjugate:
Unconjugated
Tested Applications:
IHC-P, WB
Application Note:
WB: 1-4 μg/mL; IHC-P: 1-2 μg/mL.

Antibody validated: Western Blot in human, mouse and rat samples; Immunohistochemistry in mouse, and rat samples. All other applications and species not yet tested.
Positive Control 1:
Cat. No. 1305 - Human Kidney Tissue Lysate
Positive Control 2:
Cat. No. 1405 – Mouse Kidney Tissue Lysate
Predicted Molecular Weight:
Predicted: 135 kD + 10 N-linked glycosylation sites

Observed: 200 kD
Validation:

Induced Expression Validation (Figure 10): Nephrin expression detected by anit-Nephrin antibodies was up-regulated by curcumin treatment in type 1 diabetic rats.

Localization Validation (Figure 11): Nephrin detected by anti-Nephrin antibodies is colocalized with PKGIα at the tips of the cell processes in rat podocytes.

Regulated expression validation (Figure 14): Nephrin expression detected by anit-Nephrin antibodies was down-regulated by PEGF treatment (NGP or HGP), which was reversed by the addition of C3 transferase in mouse podocyte cells.

Isoforms:
Human Nephrin has 2 isoforms, including isoform 1 (1241aa, 135kD) and isoform 2 (1201aa, 131kD). Mouse Nephrin also has 2 isoforms, including isoform 1 (1256aa, 136kD) and isoform 2 (1242aa, 135kD). Rat Nephrin has 3 isoforms, including isoform 1 (1252aa, 136kD), isoform 2 (1239aa, 135kD) and isoform 3 (1166aa, 127kD). 2265 can detect isoforms of human, mouse and rat.
Purification:
Nephrin Antibody is affinity chromatography purified via peptide column.
Clonality:
Polyclonal
Isotype:
IgG
physical-state:
Liquid
Buffer:
Nephrin Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Storage Conditions:
Nephrin antibody can be stored at 4°C up to one year. Antibodies should not be exposed to prolonged high temperatures.
Ncbi Official Symbol:
NPHS1
Additional Names:
Nephrin Antibody: CNF, NPHN, nephrin, Nephrin, Renal glomerulus-specific cell adhesion receptor
Protein Accession Number:
NP_004637
Protein Gi Number:
4758822
Ncbi Gene Id Number:
4868
User Note:
Optimal dilutions for each application to be determined by the researcher.
Background:
Nephrin Antibody: Nephrin is strongly expressed in renal glomeruli and is a member of the immunoglobulin family of cell adhesion molecules. Mutations in the Nephrin gene result in congenital nephrotic syndrome, an autosomal-recessive disorder characterized by massive proteinuria in utero and nephrosis at birth. Renal glomeruli allow normal kidneys to filter plasma so that it is very pure. Nephrin is expressed in the podocyte slit-diaphragm of the renal glomeruli in a manner that suggests that Nephrin molecules homodimerize in an anti-parallel fashion similar to cadherin interactions in adherens junctions. Thus, Nephrin may constitute the entire extracellular structure of the slit-diaphragm.
Background Reference 1:
Kestila et al. Mol. Cell 1998; 1:575-582.
Background Reference 2:
Tryggvason. J. Am. Soc. Nephrol. 1999; 10:2440-5.
Background Reference 3:
Tryggvason and Wartiovaara. Curr. Opin. Nephrol. Hypertens. 2001; 10:543-9.
Citation 1:
Chen et al. Atgl deficiency induces podocyte apoptosis and leads to glomerular filtration barrier damage. FEBS J. 2017;284(7):1070-1081. PMID: 28194887
Citation 2:
Yun et al. Expression of nephrin in the human placenta and fetal membranes. Mol Med Rep. 2015;12(4):5116-20.PMID: 26151763
Citation 3:
Li et al. Mesenchymal stem cells protect podocytes from apoptosis induced by high glucose via secretion of epithelial growth factor. Stem Cell Res Ther. 2013;4(5):103. PMID: 24004644
Citation 4:
Soetikno et al. Curcumin decreases renal triglyceride accumulation through AMPK-SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats. J Nutr Biochem. 2013;24(5):796-802. PMID: 22898567
Citation 5:
Piwkowska et al. Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type Iα subunits. Biochim Biophys Acta. 2013;1832(6):791-804.PMID: 23454089
Citation 6:
Toyama et al. Proteinuria in AMPD2-deficient mice. Genes Cells. 2012;17(1):28-38. PMID: 22212473
Citation 7:
Huang et al. Increased levels of serum pigment epithelium-derived factor aggravate proteinuria via induction of podocyte actin rearrangement. Int Urol Nephrol. 2019;51(2):359-367. PMID: 30536192
Citation 8:
Piwkowska et al. Hydrogen peroxide induces dimerization of protein kinase G type Iα subunits and increases albumin permeability in cultured rat podocytes. J Cell Physiol. 2012;227(3):1004-16. PMID: 21520075
Citation 9:
Rogacka et al. Metformin overcomes high glucose-induced insulin resistance of podocytes by pleiotropic effects on SIRT1 and AMPK. Biochim Biophys Acta Mol Basis Dis. 2018;1864(1):115-125. PMID: 29032153

FOR RESEARCH USE ONLY

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Disclaimer:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.

CATALOG NUMBER:

2265

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

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