Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A375 (human malignant melanoma epithelial cell line) cells labeling NG2 with ab255811 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in A375 cells. Negative control: SK-BR-3 (PMID: 20852124). The nuclear counterstain is DAPI (blue). Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red). The negative control is the secondary antibody only.

Immunohistochemical analysis of paraffin-embedded human melanoma tissue labeling NG2 with ab255811 at 1/100 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human melanoma (PMID: 25197555). The section was incubated with ab255811 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Flow cytometric analysis of SK-BR-3 (Human breast adenocarcinoma epithelial cell, Left panel) / A375 (Human malignant melanoma epithelial cell, Right panel) labeling NG2 with ab255811 at 1/600 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled 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. Negative control: SK-BR-3 (PMID: 20852124). Gated on viable cells.

NG2 was immunoprecipitated from 0.35 mg A375 (Human malignant melanoma epithelial cell) whole cell lysate with ab255811 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab255811 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/1000 dilution.

Lane 1: A375 whole cell lysate 10 µg (Input).
Lane 2: ab255811 IP in A375 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255811 in A375 whole cell lysate.

The molecular weight observed is consistent with what has been described in the literature (PMID: 9477982, 8790396). A truncated form is also detected (190kDa, PMID: 25387269).

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 1 second.

All lanes : Anti-NG2 antibody [EPR22410-145] (ab255811) at 1/1000 dilution

Lane 1 : A375 (human malignant melanoma epithelial cell) whole cell lysate
Lane 2 : SK-MEL-28 (human malignant melanoma) whole cell lysate
Lane 3 : SK-BR-3 (human breast adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 251 kDa
Observed band size: 330,600 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

The antibody detects NG2 core protein of 330KD and intact proteoglycan of 600KD (PMID: 9477982, 8790396). 

Negative control: SK-BR-3 (PMID: 20852124).

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling NG2 with ab255811 at 1/100 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: No staining in human colon. (PMID: 25197555). The section was incubated with ab255811 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.