Lanes 1-2 : Anti-RhoA + RhoC antibody [EPR18133] (ab187026) at 1/20000 dilution
Lane 3 : Anti-RhoA + RhoC antibody [EPR18133] (ab187026) at 1/500 dilution

Lane 1 : Human RhoA full length protein
Lane 2 : Human RhoB full length protein
Lane 3 : Human RhoC full length protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

5% NFDM/TBST: Blocking and diluting buffer.

Exposure time: Lane 1: 30 seconds; lane 2 and 3: 3 minutes.

Human RhoA full length protein (ab101594) contains aa1-193 with His-Tag® at N-Terminus; Human RhoB full length protein (ab107139) contains aa1-193 with His-Tag® at N-Terminus; Human RhoC full length protein (ab98085) contains aa1-190 with His-Tag® at N-Terminus.

The affinity of ab187016 to RhoA is higher than to RhoC.

All lanes : Anti-RhoA + RhoC antibody [EPR18133] (ab187026) at 1/2000 dilution

Lane 1 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 1 minute

5% NFDM/TBST: Blocking and diluting buffer.

All lanes : Anti-RhoA + RhoC antibody [EPR18133] (ab187026) at 1/2000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 mg/ml per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

All lanes : Anti-RhoA + RhoC antibody [EPR18133] (ab187026) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Rat brain lysate
Lane 4 : Rat kidney lysate
Lane 5 : C6 (Rat glial tumor cells) whole cell lysate
Lane 6 : Raw264.7(Mouse macrophage cells transformed with Abelson murine leukemia virus ) whole cell lysate
Lane 7 : PC12 (Rat adrenal gland pheochromocytoma ) whole cell lysate
Lane 8 : NIH 3T3 (Mouse embyro fibroblast cells ) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 10 seconds

5% NFDM/TBST: Blocking and diluting buffer.

Immunohistochemical analysis of paraffin-embedded Human transitional cell carcinoma of bladder tissue labeling RhoA + RhoC with ab187026 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic staining on cancer cells of transitional cell carcinoma of bladder is observed. Counter stained with Hematoxylin.

Negative 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 Mouse kidney tissue labeling RhoA + RhoC with ab187026 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic staining on kidney tubules of mouse kidney is observed. Counter stained with Hematoxylin.

Negative 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 stomach tissue labeling RhoA + RhoC with ab187026 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic staining on epithelial cells of rat stomach is observed. Counter stained with Hematoxylin.

Negative 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 embyro fibroblast cells) cells labeling RhoA + RhoC with ab187026 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic staining on NIH 3T3 cells is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab187026 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling RhoA + RhoC with ab187026 at 1/400 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and a unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

Immunoprecipitation of RhoA + RhoC from 1mg of NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate achieved using ab187026 at 1/50 dilution.
Lane 1: Input: 10µg of NIH/3T3 whole cell lysate.
Lane 2: NIH/3T3 whole cell lysate following IP with ab187026.
Lane 3: Negative control: IP using Rabbit monoclonal IgG (ab172730) instead of ab187026 in NIH/3T3 whole cell lysates.
Western blot was performed using ab187026 at 1/1000 dilution.
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1500 was used for detection.
Blocking and dilution buffer and concentration: 5% NFDM/TBST. 30 second exposure.