Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

All lanes : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1/5000 dilution

Lane 1 : Hela whole cell (Human)
Lane 2 : 3T3 cell (Mouse)
Lane 3 : Rat brain
Lane 4 : Xenopus embryo
Lane 5 : Chicken Liver
Lane 6 : EBTr cell (Cow)
Lane 7 : CHO cell (Chinese hamster)
Lane 8 : Pig liver

Secondary
Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L (HRP) (ab6728) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 40.2 kDa


Exposure time : 10 seconds

The membrane was blocked in 5%BSA in TBST for one hour, then incubated for one hour in primary antibody diluted in TBST.

Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 0.5 µg/ml + HeLa cell lysate

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP) (ab6789) at 1/5000 dilution
developed using the ECL technique

Performed under non-reducing conditions.

Predicted band size : 40.2 kDa


Exposure time : 30 seconds

Western blot - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

Lanes 1 - 5 : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1/1000 dilution (Blocked in 5% MILK)
Lanes 6 - 10 : Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484) at 1/1000 dilution (Blocked in 5% BSA)

Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate (ab7909)
Lane 4 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899)
Lane 5 : HEK293 Human embryonic kidney cell line Whole Cell Lysate (ab7902)
Lane 6 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 7 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 8 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate (ab7909)
Lane 9 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899)
Lane 10 : HEK293 Human embryonic kidney cell line Whole Cell Lysate (ab7902)

Lysates/proteins at 20 µg per lane.

Secondary
Goat anti-Mouse (HRP conjugated) at 1/5000 dilution

Predicted band size : 40.2 kDa
Observed band size : 40.2 kDa


The membrane 1-5 was blocked in 5% Milk (1 hour). The membrane 6-10 was blocked in 5% BSA (1 hour). Abcam routinely uses 5% BSA to block however following recent customer feedback our labs investigated the effect of 5% milk blocking. We can now confirm that milk is not a suitable blocking agent and significantly decreases the signal on the membrane. We have informed all users of the antibody of this finding.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

IHC image of GAPDH staining in human liver FFPE section, performed on a Bond^TM system using the standard protocol F. 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 ab9484, 5µg/ml, for 8 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.

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

ICC/IF image of ab9484 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab9484, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

ICC/IF image of ab9484 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab9484, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Flow Cytometry - Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

Flow Cytometry analysis of GAPDH using ab9484.

Human monocytes were fixed in paraformaldehyde and permeabilized. ab9484 was used at a 1/200 dilution. The secondary used was an Alexa-Fluor 488 conjugated chicken anti-rabbit IgG (H+L) polyclonal, used at a 1/500 dilution.

Image courtesy of Dr Mahesh Shivananjappa by Abreview.