Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/20000 dilution (Purified) + COS-1 (African green monkey  kidney fibroblast-like) whole cell lysates at 15 µg

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

Predicted band size: 36 kDa
Observed band size: 36 kDa

Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/20000 dilution (Purified) + HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 15 µg

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

Predicted band size: 36 kDa
Observed band size: 36 kDa

All lanes : Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915) at 1/10000 dilution (unpurified)

Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) cell lysate
Lane 4 : HUVEC (human umbilical vein endothelial cell line) cell lysate
Lane 5 : MCF7 (human breast adenocarcinoma cell line) cell lysate
Lane 6 : SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

Predicted band size: 36 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

Secondary antibody - anti-rabbit HRP (ab6721)

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.

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.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling GAPDH with purified ab128915 at 1/20 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 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).

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.

IHC image of ab128915 (unpurified) staining GAPDH in human pancreas* formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab128915, 1:250 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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.

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.

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.

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