All lanes : Anti-DRP1 antibody [EPR19274] (ab184247) at 1/1000 dilution

Lane 1 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 5 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 6 : HCT 116 (Human colorectal carcinoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 83 kDa
Observed band size: 83 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 3 minutes; Lane 3: 30 seconds; Lane 4,5 and 6: 8 seconds.

DRP1 can be SUMOylated, as described in the literature (PMID: 19638400).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling DRP1 with ab184247 at 1/250 dilution, followed by Goat anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasm staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR19274] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab184247 at 1/250 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291  at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling DRP1 with ab184247 at 1/70 dilution (red) compared with a Rabbit IgG,monoclonal -Isotype Control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

Anti-DRP1 antibody [EPR19274] (ab184247) at 1/1000 dilution + Human fetal kidney lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 83 kDa
Observed band size: 83 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-DRP1 antibody [EPR19274] (ab184247) at 1/1000 dilution

Lane 1 : Rat brain lysate
Lane 2 : Rat heart lysate
Lane 3 : Mouse brain lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 83 kDa
Observed band size: 83 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Lane 1: 2 seconds; Lane 2: 8 seconds; Lane 3: 3 seconds.

All lanes : Anti-DRP1 antibody [EPR19274] (ab184247) at 1/1000 dilution

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 83 kDa
Observed band size: 83 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling DRP1 with ab184247 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on mouse cerebrum is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat cerebellum tissue labeling DRP1 with ab184247 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on rat cerebellum is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling DRP1 with ab184247 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasm staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR19274] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab184247 at 1/250 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

DRP1 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab184247 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab184247 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection  at 1/10000 dilution.
Lane 1: HeLa whole cell lysate 10µg (Input).
Lane 2: ab184247 IP in HeLa whole cell lysate.
Lane 3: Rabbit IgG,monoclonal [EPR19274]-Isotype Control  (ab172730) instead of ab184247 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 5 seconds.

Note: DRP1 can be SUMOylated, as described in the literature (PMID: 19638400).