Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PARK7/DJ1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Human brain tissue lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab18257 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab18257 was shown to specifically react with PARK/DJ1 in wild-type HAP1 cells. No band was observed when PARK/DJ1 knockout samples were used. Wild-type and PARK/DJ1 knockout samples were subjected to SDS-PAGE. ab18257 and ab8245 (loading control to GAPDH) were diluted to 1µg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

All lanes : Anti-PARK7/DJ1 antibody (ab18257) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Liver (Mouse) Tissue Lysate - normal tissue
Lane 3 : Heart (Mouse) Tissue Lysate
Lane 4 : Kidney (Mouse) Tissue Lysate
Lane 5 : Mouse pancreas tissue lysate - total protein (ab29363)
Lane 6 : Testis (Mouse) Tissue Lysate - normal tissue
Lane 7 : Mouse skeletal muscle tissue lysate - total protein (ab29711)
Lane 8 : Spinal Cord (Mouse) Tissue Lysate
Lane 9 : Ovary (Mouse) Tissue Lysate - normal tissue
Lane 10 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 11 : Brain (Rat) Tissue Lysate - normal tissue
Lane 12 : Liver (Rat) Tissue Lysate
Lane 13 : Heart (Rat) Tissue Lysate
Lane 14 : Kidney (Rat) Whole Cell Lysate - normal tissue (ab29480)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 20 kDa
Observed band size: 24 kDa

why is the actual band size different from the predicted?
Additional bands at: 15 kDa. We are unsure as to the identity of these extra bands.

ICC/IF image of ab18257 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab18257, 5µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor^® 594 phalloidin was used to label F-actin (red).

All lanes : Anti-PARK7/DJ1 antibody (ab18257) at 1 µg/ml

Lane 1 : Jurkat lysate
Lane 2 : HeLa lysate
Lane 3 : 3T3 lysate
Lane 4 : Jurkat lysate with Human PARK7/DJ1 peptide (ab18659) at 1 µg/ml
Lane 5 : HeLa lysate with Human PARK7/DJ1 peptide (ab18659) at 1 µg/ml
Lane 6 : 3T3 lysate with Human PARK7/DJ1 peptide (ab18659) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG (680) at 1/10000 dilution

Predicted band size: 20 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?