Recombinant Anti-NG2 antibody [EPR23976-145] (ab275024)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR23976-145] to NG2
- Suitable for: Flow Cyt, ICC/IF, IP, WB
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-NG2 antibody [EPR23976-145]
See all NG2 primary antibodies -
Description
Rabbit monoclonal [EPR23976-145] to NG2 -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt, ICC/IF, IP, WBmore details
Unsuitable for: IHC-P -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Human pancreas tissue lysate; Mouse brain and pancreas tissue lysates; rat brain tissue lysate; A375 and SK-MEL-28 whole cell lysates. ICC/IF: Mouse primary neural/glia cells; Rat primary neural/glia cells; LADMAC cells. Flow cyt: LADMAC cells; Mouse primary neural glia cells, A-375 cells. IP: Mouse brain tissue lysate.
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General notes
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.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR23976-145 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab275024 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt |
1/500.
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ICC/IF |
1/50.
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IP |
1/30.
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WB |
1/1000. Detects a band of approximately 280, 330 kDa (predicted molecular weight: 251 kDa).
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Notes |
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Flow Cyt
1/500. |
ICC/IF
1/50. |
IP
1/30. |
WB
1/1000. Detects a band of approximately 280, 330 kDa (predicted molecular weight: 251 kDa). |
Target
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Function
Proteoglycan playing a role in cell proliferation and migration which stimulates endothelial cells motility during microvascular morphogenesis. May also inhibit neurite outgrowth and growth cone collapse during axon regeneration. Cell surface receptor for collagen alpha 2(VI) which may confer cells ability to migrate on that substrate. Binds through its extracellular N-terminus growth factors, extracellular matrix proteases modulating their activity. May regulate MPP16-dependent degradation and invasion of type I collagen participating in melanoma cells invasion properties. May modulate the plasminogen system by enhancing plasminogen activation and inhibiting angiostatin. Functions also as a signal transducing protein by binding through its cytoplasmic C-terminus scaffolding and signaling proteins. May promote retraction fiber formation and cell polarization through Rho GTPase activation. May stimulate alpha-4, beta-1 integrin-mediated adhesion and spreading by recruiting and activating a signaling cascade through CDC42, ACK1 and BCAR1. May activate FAK and ERK1/ERK2 signaling cascades. -
Tissue specificity
Detected only in malignant melanoma cells. -
Sequence similarities
Contains 15 CSPG (NG2) repeats.
Contains 2 laminin G-like domains. -
Post-translational
modificationsO-glycosylated; contains glycosaminoglycan chondroitin sulfate which are required for proper localization and function in stress fiber formation (By similarity). Involved in interaction with MMP16 and ITGA4.
Phosphorylation by PRKCA regulates its subcellular location and function in cell motility. -
Cellular localization
Apical cell membrane. Cell projection > lamellipodium membrane. Localized at the apical plasma membrane it relocalizes to the lamellipodia of astrocytoma upon phosphorylation by PRKCA. Localizes to the retraction fibers. Localizes to the plasma membrane of oligodendrocytes. - Information by UniProt
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Database links
- Entrez Gene: 1464 Human
- Entrez Gene: 121021 Mouse
- Entrez Gene: 81651 Rat
- Omim: 601172 Human
- SwissProt: Q6UVK1 Human
- SwissProt: Q8VHY0 Mouse
- SwissProt: Q00657 Rat
- Unigene: 513044 Human
see all -
Alternative names
- 4732461B14Rik antibody
- AN2 antibody
- AN2 proteoglycan antibody
see all
Images
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Flow cytometry overlay histogram showing left A-375 positive cells and right negative MCF7 stained with ab275024 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interactionfollowed by the antibody (ab275024) (1x 106 in 100μl at 5.0 μg/ml (1/402)) for 30min on ice.
The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice
Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
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ab275024 staining NG2 in A375 cells, with negative expression in MCF7 cells. The cells were fixed with 100% methanol (5 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab275024 at 5 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 µg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.
This product also work with 4% formaldehyde (10 min) fixation under the same testing conditions.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse primary neural/glia cell cells labelling NG2 with ab275024 at 1/100 (4.67 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing positive staining in mouse primary glia cells. Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection. is observed. ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain MAP2 at 1/500 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Red). The Nuclear counterstain was DAPI (Blue).
ve control 1: ab275024 at 1/100 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution.
-ve control 2: ab11267 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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All lanes : Anti-NG2 antibody [EPR23976-145] (ab275024) at 1/1000 dilution
Lane 1 : Mouse brain tissue lysate at 20 µg
Lane 2 : Mouse liver tissue lysate at 20 µg
Lane 3 : Mouse pancreas tissue lysate at 40 µg
Lane 4 : Rat brain tissue lysate at 40 µg
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution
Predicted band size: 251 kDa
Observed band size: 280,330 kDa why is the actual band size different from the predicted?Blocking and dilution buffer: 5% NFDM/TBST.
Exposure times: Lanes 1-3: 59 seconds; Lane 4: 81 seconds.
The band of 330KDa represents the intact NG2 proteoglycan modified by chondroitin sulfate, the band of 280KDa represents NG2 core protein.
The molecular weight observed is consistent with what has been described in the literature (PMID: 20455858, 16625365, 23481707).Negative control: Mouse liver (PMID: 23481707).
Samples are non-boiled as boiling may cause protein aggregates.
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Flow cytometric analysis of Mouse primary neural glia cell cells labelling NG2 with ab275024 at 1/500 dilution (0.1ug) (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left).
Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
Gated on viable cells.
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NG2 was immunoprecipitated from 0.35 mg Mouse brain tissue lysate with ab275024 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275024 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/1000 dilution.
Lane 1: Mouse brain tissue lysate 10 ug
Lane 2: ab275024 IP in Mouse brain tissue lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab275024 in mouse brain tissue lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 15 seconds.
Sample loaded onto lane 1 was non-boiled as boiling may cause protein aggregates.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized LADMAC cells labelling NG2 with ab275024 at 1/50 (9.34 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in LADMAC cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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All lanes : Anti-NG2 antibody [EPR23976-145] (ab275024) at 1/1000 dilution
Lane 1 : A375 (human malignant melanoma epithelial cell) whole cell lysate at 20 µg
Lane 2 : SK-MEL-28 (human malignant melanoma) whole cell lysate at 20 µg
Lane 3 : Human pancreas tissue lysate at 40 µg
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution
Predicted band size: 251 kDa
Observed band size: 280,330 kDa why is the actual band size different from the predicted?Blocking and dilution buffer: 5% NFDM/TBST.
Exposure times: Lane 1: 26 seconds; Lane 2: 59 seconds; Lane 3: 125 seconds.
The band of 330KDa represents the intact NG2 proteoglycan modified by chondroitin sulfate, the band of 280KDa represents NG2 core protein.
The molecular weight observed is consistent with what has been described in the literature (PMID: 20455858, 16625365, 23481707).
Samples are non-boiled as boiling may cause protein aggregates.
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Flow cytometric analysis of LADMAC (Mouse bone marrow monocyte macrophage) cells labelling NG2 with ab275024 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
Gated on viable cells.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized rat primary neural/glia cell cells labelling NG2 with ab275024 at 1/100 (4.67 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing positive staining in rat primary glia cells.Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection. ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain MAP2 at 1/500 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Red). The Nuclear counterstain was DAPI (Blue).
-ve control 1: ab275024 at 1/100 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution.
-ve control 2: ab11267 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (2)
ab275024 has been referenced in 2 publications.
- Lan G et al. Astrocytic VEGFA: An essential mediator in blood-brain-barrier disruption in Parkinson's disease. Glia 70:337-353 (2022). PubMed: 34713920
- Yang SH et al. Effects of Cadmium Exposure on Leydig Cells and Blood Vessels in Mouse Testis. Int J Environ Res Public Health 19:N/A (2022). PubMed: 35206604