Western blot - PARK7/DJ1 antibody (ab18257)

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

Lysates/proteins at 20 µg per lane.

Secondary
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?)

Immunocytochemistry/ Immunofluorescence - PARK7/DJ1 antibody (ab18257)

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).

Western blot - PARK7/DJ1 antibody (ab18257)

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 : Pancreas (Mouse) Tissue Lysate (ab29363)
Lane 6 : Testis (Mouse) Tissue Lysate - normal tissue
Lane 7 : Skeletal Muscle (Mouse) Tissue Lysate (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
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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - PARK7/DJ1 antibody (ab18257)

Image courtesy of Human Protein Atlas

ab18257 staining PARK7/DJ1 in Human parathyroid. The paraffin embedded  tissue was incubated with ab18257 (1/22000 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. ab18257 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.

Further results for this antibody can be found at www.proteinatlas.org

Western blot

Anti-PARK7/DJ1 antibody (ab18257) at 1 µg/ml + PARK7/DJ1 protein (His tag) (ab51198) at 0.01 µg

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 1 minute