Anti-eNOS antibody [M221] (ab76198)
Key features and details
- Mouse monoclonal [M221] to eNOS
- Suitable for: IHC-P, ICC/IF, Flow Cyt, WB
- Reacts with: Mouse, Human
- Isotype: IgG1
Overview
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Product name
Anti-eNOS antibody [M221]
See all eNOS primary antibodies -
Description
Mouse monoclonal [M221] to eNOS -
Host species
Mouse -
Specificity
ab76198 is not predicted to react with other NOS family members due to low homology. -
Tested Applications & Species
See all applications and species dataApplication Species Flow Cyt HumanICC/IF HumanIHC-P HumanWB MouseHuman -
Immunogen
Recombinant fragment corresponding to Mouse eNOS aa 1024-1202 (C terminal).
Database link: P70313 -
Positive control
- Human umbilical vein endothelial cells untreated and treated with lambda phosphatase. Mouse placenta lysate. Huvec lysate. IHC-P: FFPE human normal placenta tissue sections.
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General notes
This antibody detects eNOS in mouse and rat but at a lower intensity than in human. If you are working in mouse or rat, we would recommend using no more than 1% milk as the blocking agent for optimal signal strength.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at -20°C. Stable for 12 months at -20°C. -
Storage buffer
Preservative: 0.05% Sodium azide
Constituents: 0.1% BSA, 50% Glycerol, PBS -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
M221 -
Isotype
IgG1 -
Research areas
Images
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All lanes : Anti-eNOS antibody [M221] (ab76198) at 1/500 dilution
Lane 1 : Huvec cell lysates
Lane 2 : Mouse placenta lysates
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat anti Mouse IR680 at 1/10000 dilution
Predicted band size: 133 kDaThis blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab76198 overnight at 4°C. Antibody binding was detected using Goat anti Mouse IR680 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
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IHC image of eNOS staining in human normal placenta*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab76198, 1/1000 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
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ab76198 staining eNOS in human umbilical vein endothelial cells. Cells with fixed with paraformaldehyde.
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Overlay histogram showing HEK293 cells stained with ab76198 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76198, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
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All lanes : Anti-eNOS antibody [M221] (ab76198) at 1/1000 dilution
Lane 1 : human umbilical vein endothelial
cells, untreated
Lane 2 : human umbilical vein endothelial
cells, treated with lambda phosphatase
Predicted band size: 133 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?