All lanes : Anti-VCP antibody [EPR3307(2)] (ab109240) at 1/10000 dilution (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : A549 (Human lung carcinoma epithelial cell) whole cell lysates
Lane 3 : C6 (Rat glial tumor glial cell) whole cell lysates)
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 5 : RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 6 : SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 89 kDa
Observed band size: 89 kDa

Blocking and diluting buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling VCP with purified ab109240 at 1:250 dilution (1.4 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunofluorescent staining of HeLa cells using unpurified ab109240 at 1/100 dilution.

ab109240 (purified) at 1:20 dilution (2ug) immunoprecipitating VCP in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2 (+): ab109240 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109240 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of HL-60 (Human acute promyelocytic leukemia promyeloblast) cells labeling VCP with purified ab109240 at 1:300 dilution (1 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - 90% methanol. Unlabeled control - Rabbit monoclonal IgG (Black).Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of MCF-7 (Human breast adenocarcinoma epithelial cell) cells labeling VCP with Purified ab109240 at 1:500 dilution (0.7μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using unpurified ab109240 at 1/100 dilution.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

All lanes : Anti-VCP antibody [EPR3307(2)] (ab109240) at 1/10000 dilution (unpurified)

Lane 1 : MCF7 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : A549 cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 10 µg/ml per lane.

Predicted band size: 89 kDa

Overlay histogram showing HL60 cells stained with  unpurified ab109240 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109240, 1/100) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HL60 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.