Anti-GAPDH antibody [EPR16891] - BSA and Azide free (ab199553) at 1/100000 dilution + HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates at 15 µg

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

Predicted band size: 36 kDa

Blocking and Diluting buffer and concentration:5% NFDM/TBST

Exposure time:3 seconds

Immunocytochemistry/immunofluorescence staining of 4% paraformaldehyde fixed; 0.1% triton X 100 permeabilized HeLa (human cervix adenocarcinoma) cells labeling GAPDH with ab181602 at dilution of 1/500. The secondary antibody used was Alexa Fluor^® 488; goat anti-rabbit IgG (ab150077) at a dilution of 1/400. Nucleus was counter-stained with DAPI (blue). ab7291, a mouse anti-tubulin antibody (1/500) was used to stain tubulin along with ab150120 (AlexaFluor^®594 goat anti-mouse secondary, 1/500). The negative controls are shown in the bottom middle and right hand panels- for negative control 1 primary antibody (ab181602; 1/500) and secondary antibody (ab150120; 1/500) was used. For negative control 2 primary antibody (ab7291; 1/500) and secondary antibody (ab150077; 1/400) was used.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181602).

Immunohistochemical analysis of paraffin-embedded human transitional cell carcinoma of bladder tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasmic and nucleus staining on the tumor cells of transitional cell carcinoma of human bladder is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181602).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasmic staining on lymphocytes of mouse spleen is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181602).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasmic staining on lymphocyte of rat spleen is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181602).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Clone EPR16891 (ab199553) has been successfully conjugated by Abcam. This image was generated using Anti-GAPDH antibody [EPR16891] (Alexa Fluor® 488). Please refer to ab201768 for protocol details.

ab201768 staining GAPDH in HeLa cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab201768 at 1/200 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Clone EPR16891 (ab199553) has been successfully conjugated by Abcam. This image was generated using Anti-GAPDH antibody [EPR16891] (Alexa Fluor® 647). Please refer to ab201272 for protocol details.

ab201272 staining GAPDH in HeLa cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab201272 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 2µg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Clone EPR16891 (ab199553) has been successfully conjugated by Abcam. This image was generated using Anti-GAPDH antibody [EPR16891] (PE). Please refer to ab224004 for protocol details.

Overlay histogram showing HeLa cells stained with ab224004 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab224004, 1/1000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

This antibody gave a positive signal in HeLa cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling GAPDH with ab181602 at 1/180 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181602).

GAPDH was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab181602 at 1/60 dilution. Western blot was performed from the immunoprecipitate using ab181602 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1: HeLa whole cell extract.

Lane 2: PBS instead of HeLa whole cell extract.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181602).