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.

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.

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.

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.

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.