All lanes : Anti-PMP70 antibody (ab85550) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : ABCD3 (PMP70) knockout HAP1 whole cell lysate
Lane 3 : A431 whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 75 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab85550 observed at 75 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab85550 was shown to specifically react with PMP70 in wild-type HAP1 cells as signal was lost in ABCD3 (PMP70) knockout cells. Wild-type and ABCD3 (PMP70) knockout samples were subjected to SDS-PAGE. Ab85550 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab85550 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab85550, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit 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. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2 cells at 1µg/ml.

All lanes : Anti-PMP70 antibody (ab85550) at 1 µg/ml

Lane 1 : Liver (Rat) Tissue Lysate
Lane 2 : Kidney (Rat) Tissue Lysate
Lane 3 : Human liver tissue lysate - total protein (ab29889)
Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 75 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?
Additional bands at: 25 kDa, 28 kDa, 37 kDa, 50 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 2 minutes


The 73 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to Rat ATP-binding cassette sub-family D member 3 (PMP70).

IHC image of Ab85550 staining in Human Cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica 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 Ab85550, 1µg/ml, 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.

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.

Anti-PMP70 antibody (ab85550) at 1 µg/ml + Lung (Mouse) Tissue Lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 75 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute