Anti-GIV antibody [EPR18433] (ab179481) at 1/5000 dilution + HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

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

Predicted band size: 216 kDa
Observed band size: 208-216 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Multiple bands represent 5 isoforms ranging from 208 to 216 kDa (PMID: 23195430; PMID: 22308453).

GIV was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab179481 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab179481 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: ab179481 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab179481 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 seconds.

Anti-GIV antibody [EPR18433] (ab179481) at 1/1000 dilution + Human fetal kidney lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 216 kDa
Observed band size: 208-216 kDa why is the actual band size different from the predicted?


Exposure time: 2 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Multiple bands represent 5 isoforms ranging from 208 to 216 kDa (PMID: 23195430; PMID: 22308453).

Anti-GIV antibody [EPR18433] (ab179481) at 1/1000 dilution + HCT 116 (Human colorectal carcinoma cell line) whole cell lysate at 20 µg

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

Predicted band size: 216 kDa
Observed band size: 208-216 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Multiple bands represent 5 isoforms ranging from 208 to 216 kDa (PMID: 23195430; PMID: 22308453).

All lanes : Anti-GIV antibody [EPR18433] (ab179481) at 1/5000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat 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: 216 kDa
Observed band size: 208-216 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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

Based on UniProt annotation, mouse GIV has 3 isoforms (215, 212, 205 kDa).

All lanes : Anti-GIV antibody [EPR18433] (ab179481) at 1/5000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma 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: 216 kDa
Observed band size: 208-216 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Based on UniProt annotation, mouse GIV has 3 isoforms (215, 212, 205KD).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling GIV with ab179481 at 1/2000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with  Anti-alpha Tubulin antibody-Loading Control  (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (AlexaFluor®594) preadsorbed (ab150120) at  1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab179481 at 1/2000 dilution followed by ab150120 at 1/1000 dilution.
-ve control 2: ab7291  at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling GIV with ab179481 at 1/2000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and membranous staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody-Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L  (AlexaFluor®594) preadsorbed (ab150120) at  1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab179481 at 1/2000 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 HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling GIV with ab179481 at 1/150 dilution (red) compared with a rabbit monoclonal IgG 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.