Anti-VCP antibody [EPR3307(2)]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-VCP antibody [EPR3307(2)] (AB109240)

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 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCP antibody [EPR3307(2)] (AB109240)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCP antibody [EPR3307(2)] (AB109240)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCP antibody [EPR3307(2)] (AB109240)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-VCP antibody [EPR3307(2)] (AB109240)

Intracellular 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 Fluorr^® 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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-VCP antibody [EPR3307(2)] (AB109240)

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

• IP

Lab

Immunoprecipitation - Anti-VCP antibody [EPR3307(2)] (AB109240)

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.

All lanes:

Immunoprecipitation - Anti-VCP antibody [EPR3307(2)] (ab109240)

Predicted band size: 89 kDa

Observed band size: 89 kDa

false

• WB

Lab

Western blot - Anti-VCP antibody [EPR3307(2)] (AB109240)

ab109240 was shown to react with VCP in wild-type U2OS cells in Western blot with loss of signal observed in a VCP siRNA knockdown cell line. Cell lysates from wild-type U2OS transfected with either scrambled siRNA or VCP siRNA were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab109240 overnight at 4 °C at a 1/50000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-VCP antibody [EPR3307(2)] (ab109240) at 1/50000 dilution

Lane 1:

Wild-type U2OS transfected with scrambled siRNA control lysate at 10 µg

Lane 2:

U2OS transfected with siRNA specifically targeting VCP cell lysate at 10 µg

false

• WB

Lab

Western blot - Anti-VCP antibody [EPR3307(2)] (AB109240)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-VCP antibody [EPR3307(2)] (ab109240) at 1/10000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

C6 (Rat glial tumor glial cell) whole cell lysates) at 20 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 5:

RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Lane 6:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates 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 1/20000 dilution

Predicted band size: 112 kDa,130 kDa,151 kDa,217 kDa,43 kDa,469 kDa,47 kDa,60 kDa,65 kDa,89 kDa,92 kDa

Observed band size: 89 kDa

false

• WB

Unknown

Western blot - Anti-VCP antibody [EPR3307(2)] (AB109240)

All lanes:

Western blot - Anti-VCP antibody [EPR3307(2)] (ab109240) at 1/10000 dilution

Lane 1:

MCF7 cell lysate at 10 µg/mL

Lane 2:

HeLa cell lysate at 10 µg/mL

Lane 3:

A549 cell lysate at 10 µg/mL

Lane 4:

SH-SY5Y cell lysate at 10 µg/mL

Predicted band size: 89 kDa

false