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

Lane 1 : HeLa Whole Cell Lysate
Lane 2 : NIH 3T3 Whole Cell Lysate
Lane 3 : PC12 Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 36 kDa


Exposure time: 1 minute

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab9484 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

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.

Overlay histogram showing HeLa cells stained with ab9484 (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 (ab9484, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was goat anti-mouse DyLight^® 488 (IgG H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

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 laevis embryo
Lane 5 : Chicken Liver
Lane 6 : EBTr cell (Cow)
Lane 7 : CHO cell (Chinese hamster)
Lane 8 : Pig liver

Secondary
All lanes : Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

The membrane was blocked in 5% BSA in TBST for 1 hour, then incubated for 1 hour in primary antibody diluted in TBST.

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)

Lanes 1 & 6 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lanes 2 & 7 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lanes 3 & 8 : A-431 whole cell lysate (ab7909)
Lanes 4 & 9 : Jurkat whole cell lysate (ab7899)
Lanes 5 & 10 : HEK-293 whole cell lysate (ab7902)

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-5 : Goat anti-Mouse (HRP conjugated) at 1/5000 dilution
Lanes 6-10 : Goat anti-Mouse (HRP conjugated) at 1/5000 dilution

Predicted band size: 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

The membrane 1-5 was blocked in 5% milk (1 hour). The membrane 6-10 was blocked in 5% BSA (1 hour). Milk is not a suitable blocking agent and significantly decreases the signal on the membrane.

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

Secondary
Goat Anti-Mouse IgG H&L (HRP) (ab6789) at 1/5000 dilution

Developed using the ECL technique.

Performed under non-reducing conditions.

Predicted band size: 36 kDa


Exposure time: 30 seconds