Recombinant Anti-NG2 antibody [EPR23976-145] (ab275024)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.