Anti-PODXL 抗体 [EPR9518] (ab150358)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR9518] to PODXL
- Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
製品の概要
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製品名
Anti-PODXL antibody [EPR9518]
PODXL 一次抗体 製品一覧 -
製品の詳細
Rabbit monoclonal [EPR9518] to PODXL -
由来種
Rabbit -
アプリケーション
適用あり: Flow Cyt (Intra), WB, IHC-P, ICC/IFmore details
適用なし: IP -
種交差性
交差種: Human -
免疫原
Recombinant fragment within Human PODXL aa 300-500. The exact sequence is proprietary.
Database link: O00592 -
ポジティブ・コントロール
- WB: Raji, HeLa, HCT116 and HAP1 whole cell lysate. Human fetal kidney lysate. IHC-P: Human kidney tissue. Human hepatocellular carcinoma, breast carcinoma and endometrial carcinoma tissue. Human glioma tissue. ICC/IF: HeLa cells. Flow Cyt (intra): HeLa cells.
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特記事項
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
製品の特性
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製品の状態
Liquid -
保存方法
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
バッファー
Preservative: 0.01% Sodium azide
Constituents: 0.05% BSA, 40% Glycerol, PBS -
Concentration information loading...
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精製度
Protein A purified -
ポリ/モノ
モノクローナル -
クローン名
EPR9518 -
アイソタイプ
IgG -
研究分野
関連製品
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Alternative Versions
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
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Recombinant Protein
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab150358の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
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Flow Cyt (Intra) |
1/250.
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WB |
1/1000 - 1/10000. Detects a band of approximately 165 kDa (predicted molecular weight: 58 kDa).
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IHC-P | (2) |
1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
For unpurified use at 1/250 - 1/500. |
ICC/IF | (1) |
1/100.
For unpurified use at 1/500. |
特記事項 |
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Flow Cyt (Intra)
1/250. |
WB
1/1000 - 1/10000. Detects a band of approximately 165 kDa (predicted molecular weight: 58 kDa). |
IHC-P
1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. For unpurified use at 1/250 - 1/500. |
ICC/IF
1/100. For unpurified use at 1/500. |
ターゲット情報
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機能
Involved in the regulation of both adhesion and cell morphology and cancer progression. Function as an anti-adhesive molecule that maintains an open filtration pathway between neighboring foot processes in the podocyte by charge repulsion. Acts as a pro-adhesive molecule, enhancing the adherence of cells to immobilized ligands, increasing the rate of migration and cell-cell contacts in an integrin-dependent manner. Induces the formation of apical actin-dependent microvilli. Involved in the formation of a preapical plasma membrane subdomain to set up inital epithelial polarization and the apical lumen formation during renal tubulogenesis. Plays a role in cancer development and aggressiveness by inducing cell migration and invasion through its interaction with the actin-binding protein EZR. Affects EZR-dependent signaling events, leading to increased activities of the MAPK and PI3K pathways in cancer cells. -
組織特異性
Glomerular epithelium cell (podocyte). -
配列類似性
Belongs to the podocalyxin family. -
ドメイン
Both the O-glycan-rich domain of the extracellular domain and the C-terminus PDZ-binding motif (DTHL) in the cytoplasmic tail harbor an apical sorting signal. The cytoplasmic domain is necessary for the apical membrane targeting and renal tubulogenesis. The cytoplasmic C-terminus PDZ-binding motif (DTHL) is essential for interaction with SLC9A3R1 and for targeting SLC9A3R1 to the apical cell membrane. The extracellular domain is necessary for microvillus formation (By similarity). The large highly anionic extracellular domain allows to maintain open filtration pathways between neighboring podocyte foot processes. -
翻訳後修飾
N- and O-linked glycosylated. Sialoglycoprotein. -
細胞内局在
Apical cell membrane. Cell projection, lamellipodium. Cell projection, filopodium. Cell projection, ruffle. Cell projection, microvillus. Membrane raft. Membrane. In single attached epithelial cells is restricted to a preapical pole on the free plasma membrane whereas other apical and basolateral proteins are not yet polarized. Colocalizes with SLC9A3R2 at the apical plasma membrane during epithelial polarization. Colocalizes with SLC9A3R1 at the trans-Golgi network (transiently) and at the apical plasma membrane. Its association with the membrane raft is transient. Colocalizes with actin filaments, EZR and SLC9A3R1 in a punctate pattern at the apical cell surface where microvilli form. Colocalizes with EZR and SLC9A3R2 at the apical cell membrane of glomerular epithelium cells (By similarity). Forms granular, punctuated pattern, forming patches, preferentially adopting a polar distribution, located on the migrating poles of the cell or forming clusters along the terminal ends of filipodia establishing contact with the endothelial cells. Colocalizes with the submembrane actin of lamellipodia, particularly associated with ruffles. Colocalizes with vinculin at protrusions of cells. Colocalizes with ITGB1. Colocalizes with PARD3, PRKCI, EXOC5, OCLN, RAB11A and RAB8A in apical membrane initiation sites (AMIS) during the generation of apical surface and luminogenesis (By similarity). - Information by UniProt
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参照データベース
- Entrez Gene: 5420 Human
- Omim: 602632 Human
- SwissProt: O00592 Human
- Unigene: 744213 Human
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製品の状態
There are 2 isoforms produced by alternative splicing. -
別名
- GCTM-2 antigen antibody
- Gp2 antibody
- Gp200 antibody
see all
画像
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Flow cytometry overlay histogram showing wild-type Hela (green line) and PODXL knockout Hela stained with ab150358 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab150358) (1x 106 in 100μl at 1.0 μg/ml (1/2250)) for 30min at 22°C.
The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hela - black line, PODXL knockout Hela - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
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All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/10000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PODXL knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 58 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab150358 observed at 160 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab150358 was shown to react with PODXL in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264984 (knockout cell lysate ab257210) was used. Wild-type HeLa and PODXL knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab150358 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling PODXL with purified ab150358 at 1/1000 dilution (0.44 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) is the secondary antibody.
PBS instead of the primary antibody was used as the negative control.
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All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : PODXL knockout HCT116 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 58 kDa
Observed band size: 200 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab150358 observed at 200 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.
ab150358 was shown to react with PODXL in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266887 (knockout cell lysate ab257211) was used. Wild-Type HCT116 and PODXL knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab150358 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PODXL knockout HAP1 whole cell lysate
Lane 3 : Raji whole cell lysate
Lane 4 : HeLa whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 58 kDaLanes 1 - 4: Merged signal (red and green). Green - ab150358 observed at 160 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab150358 was shown to specifically react with PODXL in wild-type cells as signal was lost in PODXL knockout cells. Wild-type and PODXL knockout samples were subjected to SDS-PAGE. Ab150358 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
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Anti-PODXL antibody [EPR9518] (ab150358) at 1/10000 dilution (purified) + Human fetal kidney lysates at 15 µg
Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution
Predicted band size: 58 kDa
Observed band size: 165 kDa why is the actual band size different from the predicted?Blocking and diluting buffer: 5% NFDM/TBST.
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Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labeling PODXL with purified ab150358 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue).
Secondary Only Control: PBS was used instead of the primary antibody as the negative control.
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All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution (unpurified)
Lane 1 : Raji lysate
Lane 2 : HeLa lysate
Lane 3 : Human fetal kidney lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution
Predicted band size: 58 kDa
Observed band size: 165 kDa why is the actual band size different from the predicted? -
Immunohistochemical analysis of paraffin embedded human kidney tissue labeling PODXL with unpurified ab150358 antibody at a dilution of 1/100. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.
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Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling PODXL with purified ab150358 at 1/250 dilution (red). The secondary antibody was Goat anti rabbit IgG (Alexa Fluor® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.
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Unpurified ab150358 staining PODXL in human kidney tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue samples were fixed with formaldehyde, cut into 20 micron slices, permeabilized with 0.05% tween-20 and blocked for 60 minutes at 25°C. Antigen retrieval was by heat mediation. The sample was incubated with primary antibody at a dilution of 1/250 at 25°C for 1 hour. An Alexa Fluor® 488-conjugated donkey anti-rabbit polyclonal (1/1000) was used as the secondary antibody, at a dilution of 1/1200.
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Immunohistochemical analysis of paraffin embedded human hepatocellular carcinoma vessels using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.
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Immunohistochemical analysis of paraffin embedded human breast adenocarcinoma tissue using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.
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Immunohistochemical analysis of paraffin embedded human endometrial carcinoma tissue using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.
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Immunohistochemical analysis of paraffin embedded human skeletal muscle tissue using unpurified ab150358 showing negative staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.
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Immunohistochemical analysis of paraffin embedded normal human kidney tissue using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.
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Immunohistochemical analysis of paraffin embedded human glioma tissue using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.
プロトコール
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (12)
ab150358 は 12 報の論文で使用されています。
- Nagano H et al. Knockdown of Podocalyxin Post-Transcriptionally Induces the Expression and Activity of ABCB1/MDR1 in Human Brain Microvascular Endothelial Cells. J Pharm Sci 111:1812-1819 (2022). PubMed: 35182544
- Yu X et al. Maturation of nephrons by implanting hPSC-derived kidney progenitors under kidney capsules of unilaterally nephrectomized mice. Curr Stem Cell Res Ther N/A:N/A (2022). PubMed: 35984016
- Dhondt B et al. Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. J Extracell Vesicles 9:1736935 (2020). PubMed: 32284825
- Zhai S et al. IL-17 aggravates renal injury by promoting podocyte injury in children with primary nephrotic syndrome. Exp Ther Med 20:409-417 (2020). PubMed: 32537005
- Ito S et al. Identification of Cell-Surface Proteins Endocytosed by Human Brain Microvascular Endothelial Cells In Vitro. Pharmaceutics 12:N/A (2020). PubMed: 32585920