ProSci

Nephrin Antibody

Product Code:
 
PSI-2265
Product Group:
 
Primary Antibodies
Supplier:
 
ProSci
Host Type:
 
Rabbit
Antibody Isotype:
 
IgG
Antibody Clonality:
 
Polyclonal
Regulatory Status:
 
RUO
Applications:
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Immunofluorescence (IF)
  • Immunohistochemistry (IHC)
  • Western Blot (WB)
1 / 14
<strong>Figure 1 Western Blot Validation in Mouse Kidney Tissue Lysate with the (A) absence or the (B) presence of blocking peptide</strong><br> Loading: 15 μg of lysates per lane. Antibodies: : Nephrin 2265 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
2 / 14
<strong>Figure 2 Western Blot Validation in Human and Mouse Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: Nephrin 2265 (2 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
3 / 14
<strong>Figure 3 Immunohistochemistry Validation of Nephrin in Mouse Kidney Tissue</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.
4 / 14
<strong>Figure 4 Immunofluorescence Validation of Nephrin in Mouse Kidney Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse kidney cells labeling Nephrin with 2265 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
5 / 14
<strong>Figure 5 Immunohistochemistry Validation of Nephrin in Rat Kidney Tissue</strong><br> Immunohistochemical analysis of paraffin-embedded rat kidney tissue using anti- Nephrin antibody (2265) at 5 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.
6 / 14
<strong>Figure 6 Immunofluorescence Validation of Nephrin in Rat Kidney Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed rat kidney tissue labeling Nephrin with 2265 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
7 / 14
<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.
8 / 14
<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).
9 / 14
<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.
10 / 14
<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 decrease in nephrin protein expression was markedly increased by curcumin treatment (P<.05) to near-normal levels. (n=5 in each group)
11 / 14
<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.
12 / 14
<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.
13 / 14
<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 and AMPD2-deficient mice at any age was observed.
14 / 14
<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.
❮❯

<strong>Figure 1 Western Blot Validation in Mouse Kidney Tissue Lysate with the (A) absence or the (B) presence of blocking peptide</strong><br> Loading: 15 μg of lysates per lane. Antibodies: : Nephrin 2265 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 Western Blot Validation in Human and Mouse Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: Nephrin 2265 (2 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 Immunohistochemistry Validation of Nephrin in Mouse Kidney Tissue</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 4 Immunofluorescence Validation of Nephrin in Mouse Kidney Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse kidney cells labeling Nephrin with 2265 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
<strong>Figure 5 Immunohistochemistry Validation of Nephrin in Rat Kidney Tissue</strong><br> Immunohistochemical analysis of paraffin-embedded rat kidney tissue using anti- Nephrin antibody (2265) at 5 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 Immunofluorescence Validation of Nephrin in Rat Kidney Tissue</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed rat kidney tissue labeling Nephrin with 2265 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
<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 decrease in 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 and AMPD2-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.

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Further Information

Additional Names:
Nephrin Antibody: CNF, NPHN, nephrin, Nephrin, Renal glomerulus-specific cell adhesion receptor
Application Note:
WB: 1-2 μg/mL; IHC: 1-5 μg/mL; IF: 10-20 μg/mL.

Antibody validated: Western Blot in human, mouse and rat samples; Immunohistochemistry in mouse and rat samples; Immunofluorescence in human, mouse and rat samples. All other applications and species not yet tested.
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 References:
  • Kestila et al. Mol. Cell 1998; 1:575-582.
  • Tryggvason. J. Am. Soc. Nephrol. 1999; 10:2440-5.
  • Tryggvason and Wartiovaara. Curr. Opin. Nephrol. Hypertens. 2001; 10:543-9.
Buffer:
Nephrin Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Conjugate:
Unconjugated
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.
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.
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.
NCBI Gene ID #:
4868
NCBI Official Name:
nephrosis 1, congenital, Finnish type (nephrin)
NCBI Official Symbol:
NPHS1
NCBI Organism:
Homo sapiens
Physical State:
Liquid
Protein Accession #:
NP_004637
Protein GI Number:
4758822
Purification:
Nephrin Antibody is affinity chromatography purified via peptide column.
Research Area:
Homeostasis
Swissprot #:
O60500
User NOte:
Optimal dilutions for each application to be determined by the researcher.
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.

References

  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
  2. Yun et al. Expression of nephrin in the human placenta and fetal membranes. Mol Med Rep. 2015;12(4):5116-20.PMID: 26151763
  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
  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
  5. Piwkowska et al. Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type I? subunits. Biochim Biophys Acta. 2013;1832(6):791-804.PMID: 23454089
  6. Toyama et al. Proteinuria in AMPD2-deficient mice. Genes Cells. 2012;17(1):28-38. PMID: 22212473
  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
  8. Piwkowska et al. Hydrogen peroxide induces dimerization of protein kinase G type I? subunits and increases albumin permeability in cultured rat podocytes. J Cell Physiol. 2012;227(3):1004-16. PMID: 21520075
  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