Anti-GAPDH antibody [EPR6256] - Loading Control

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab128915 (unpurified) (red line). The cells were fixed with 80% methanol (5 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 (ab128915, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was goat anti-rabbit Alexa Fluor^® 488 (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

ab128915 (unpurified) staining GAPDH in human HeLa (human epithelial cell line from cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody. Counter stained with DAPI.

This image is courtesy of an Abreview submitted by Kirk McManus

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling GAPDH with purified ab128915 at 1/2000 dilution (0.06 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling GAPDH with purified ab128915 at 1/20 dilution (10μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

ab128915 (unpurified) staining GAPDH in HeLa (human epithelial cell line from cervix adenocarcinoma) cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab128915 at 2μg/ml and ab7291 at 1μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an goat anti-rabbit AlexaFluor®488 (ab150081) at 2 μg/ml (shown in green) and goat anti-mouse AlexaFluor®594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Negative controls : 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

ab128915 (unpurified), at 1/250, staining GAPDH in MCF7 (human breast adenocarcinoma cell line) cells by immunofluorescence.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling GAPDH with purified ab128915 at 1/250 dilution (0.4 μg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/ml) dilution. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IP

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Immunoprecipitation - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

ab128915 (purified) at 1/20 dilution (0.5ug) immunoprecipitating GAPDH in HeLa whole cell lysates.
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates 10ug
Lane 2 (+) : ab128915 & HeLa whole cell lysates
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab128915 in HeLa whole cell lysates
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915)

Predicted band size: 25 kDa,36 kDa

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• WB

Lab

Western blot - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of HeLa whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab128915 (1/200,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab128915 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/200000 dilution

All lanes:

HeLa whole cell lysate at 15 µg with NDFM/TBST

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

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Exposure time: 60s

• WB

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Western blot - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

Secondary antibody - anti-rabbit HRP (ab6721)

All lanes:

Western blot - Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/10000 dilution

Lane 1:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate at 10 µg

Lane 2:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Lane 3:

HepG2 (human liver hepatocellular carcinoma cell line) cell lysate at 10 µg

Lane 4:

HUVEC (human umbilical vein endothelial cell line) cell lysate at 10 µg

Lane 5:

MCF7 (human breast adenocarcinoma cell line) cell lysate at 10 µg

Lane 6:

SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate at 10 µg

Secondary

All lanes:

HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

Predicted band size: 36 kDa

Observed band size: 35 kDa

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• WB

Lab

Western blot - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

All lanes:

Western blot - Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/20000 dilution

All lanes:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 15 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: 36 kDa

Observed band size: 36 kDa

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• WB

Lab

Western blot - Anti-GAPDH antibody [EPR6256] - Loading Control (AB128915)

All lanes:

Western blot - Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/20000 dilution

All lanes:

COS-1 (African green monkey  kidney fibroblast-like) whole cell lysates at 15 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: 36 kDa

Observed band size: 36 kDa

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