MA3-928

antibody from Invitrogen Antibodies

Targeting: ATP1A1

Provider product page for MA3-928

Western blot

ELISA

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [35]
Comments [0]
Validations
Western blot [4]
Immunocytochemistry [3]
Immunohistochemistry [3]
Other assay [6]

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Product number: MA3-928 - Provider product page
Provider: Invitrogen Antibodies
Product name: ATP1A1 Monoclonal Antibody (M7-PB-E9)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-928 detects sodium/potassium ATPase alpha from human, mouse, bovine, sheep, canine, chicken and porcine tissues. This antibody detects the alpha 3 isoform in rat only. MA3-928 has been successfully used in Western blot, immunohistochemistry, immunoprecipitation, immunocytochemistry, immunofluorescence, and ELISA procedures. By Western blot, this antibody detects an ~110 kDa protein representing alpha sodium/potassium ATPase from MDCK cell extract. Immunohistochemical staining of alpha sodium/potassium ATPase in porcine heart with MA3-928 yields a pattern consistent with the plasma membrane localization. The MA3-928 antigen is purified sheep kidney alpha sodium/potassium ATPase. This antibody recognizes an epitope between amino acid residues 646 and 652 of the sheep kidney alpha sodium/potassium ATPase.
Reactivity: Human, Mouse, Rat, Bovine, Canine, Chicken/Avian, Porcine
Host: Mouse
Isotype: IgG
Antibody clone number: M7-PB-E9
Vial size: 400 µL
Concentration: 1.87 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

ATP1A1 protein structure - MA3-928 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Sodiµm/Potassium ATPase alpha was performed by loading 25 µg of U251 (lane 1), human kidney (lane 2) and mouse kidney (lane 3) onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a Sodiµm/Potassium ATPase alpha monoclonal antibody (Product # MA3-928) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Images were taken at an exposure time of 2 min (lanes 1 and 3) and 4 min (lane 2). Results show a band at ~110 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: CRISPR-Cas9 mediated genome editing ofATP1A1 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR847155_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of ATP1A1 was performed by loading 30 µg of MCF7 Wild Type (Lane 1), MCF7 Cas9 (Lane 2) and MCF7 Cas9 cells transduced with ATP1A1 Lentiviral sgRNA (Lane 3) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with ATP1A1 Monoclonal Antibody (M7-PB-E9) (Product # MA3-928) using 1:1,000 dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177 1:4,000 dilution). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toATP1A1 (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of ATP1A1 was achieved by transfecting MDA-MB-231 cells with ATP1A1 specific siRNAs (Silencer® select Product # s1719). Western blot analysis (Fig. a) was performed using whole cell extracts from the ATP1A1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ATP1A1 Monoclonal Antibody (Product # MA3-928, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752) . Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to ATP1A1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-ATP1A1 Monoclonal Antibody(Product # MA3-928) and 110 kDa band corresponding to ATP1A1 was observed across the cell lines tested. Membrane Enriched extracts (30ug lysate) of Hep G2 (Lane 1), MCF-7 (Lane 2), MDA-MB-231 (Lane 3), MCF 10A (Lane 4), A-431 (Lane 5), C2C12 (Lane 6) and RAW 264.7 (Lane 7) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Sodiµm/Potassium ATPase alpha using Sodiµm/Potassium ATPase alpha Monoclonal Antibody (M7-PB-E9) (Product # MA3-928) shows staining in Hela Cells. Sodiµm/Potassium ATPase alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Sodiµm/Potassium ATPase alpha (Product # MA3-928) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Sodiµm/Potassium ATPase alpha using Sodiµm/Potassium ATPase alpha Monoclonal Antibody (M7-PB-E9) (Product # MA3-928) shows staining in MCF-7 Cells. Sodiµm/Potassium ATPase alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Sodiµm/Potassium ATPase alpha (Product # MA3-928) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Sodiµm/Potassium ATPase alpha using Sodiµm/Potassium ATPase alpha Monoclonal Antibody (M7-PB-E9) (Product # MA3-928) shows staining in U251 Cells. Sodiµm/Potassium ATPase alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Sodiµm/Potassium ATPase alpha (Product # MA3-928) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human colon carcinoma tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing Sodiµm/Potassium ATPase alpha (Product # MA3-928) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized Human testis tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Sodiµm/Potassium ATPase alpha (Product # MA3-928) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized Human tonsil tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Sodiµm/Potassium ATPase alpha (Product # MA3-928) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 3 (A) Polarized CaCo-2w cells with localization of flotillin-1 (red), Na-K-ATPase (green) and flotillin-1 and Na-K-ATPase immunofluorescence overlay (right). Flotillin-1 and Na-K-ATPase co-localize in the basolateral membrane. (B) Confocal microscopy image of polarized CaCo-2w cells. Flotillin-1 (red) is localized in a subapical compartment, but not in the apical membrane. Na-K- ATPase (green) marks the basolateral membrane.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 6 Three-dimensional, confocal-microscopic images of a human colonic epithelial layer in a human endoscopic biopsy. Flotillin-1 is marked in red, Na-K-ATPase in green, immunofluorescence overlay on the right. Na-K-ATPase was used a marker of the basolateral cell membrane.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 8 Representative western blot image for detection of membrane associated TK1 levels in plasma membrane protein extracts from the non-small cell lung carcinoma cell line NCI-H460 and healthy mono nuclear cells. a Plasma membrane protein extract from NCI-H460 had high levels of membrane associate TK1. b Membrane associated TK1 was not detected in plasma membrane proteins extracts from healthy mono nuclear cells