Anti-NG2 antibody [EPR22410-145]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NG2 antibody [EPR22410-145] (AB255811)

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.

• Flow Cyt

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Flow Cytometry - Anti-NG2 antibody [EPR22410-145] (AB255811)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NG2 antibody [EPR22410-145] (AB255811)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NG2 antibody [EPR22410-145] (AB255811)

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.

• IP

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Immunoprecipitation - Anti-NG2 antibody [EPR22410-145] (AB255811)

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:

Immunoprecipitation - Anti-NG2 antibody [EPR22410-145] (ab255811)

Predicted band size: 251 kDa

Observed band size: 190 kDa,330 kDa,600 kDa

false

• WB

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Western blot - Anti-NG2 antibody [EPR22410-145] (AB255811)

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.

All lanes:

Western blot - Anti-NG2 antibody [EPR22410-145] (ab255811) 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:

SK-BR-3 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 251 kDa

Observed band size: 330 kDa,600 kDa

false

Exposure time: 15s