Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling gamma Tubulin with ab179503 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasmic and centrosome staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
1. ab179503 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

Anti-gamma Tubulin antibody [EPR16793] (ab179503) at 1/20000 dilution + Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates at 20 µg

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

Predicted band size: 51 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-gamma Tubulin antibody [EPR16793] (ab179503) at 1/2000 dilution

Lane 1 : Human fetal brain lysates
Lane 2 : Human fetal heart lysates
Lane 3 : Human fetal kidney lysates

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 51 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-gamma Tubulin antibody [EPR16793] (ab179503) at 1/2000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : MDCK (Canine kidney cell line) whole cell lysates
Lane 3 : MDBK (Bovine kidney cell line) whole cell lysates
Lane 4 : COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysates
Lane 5 : Zebrafish lysates
Lane 6 : Xenopus (X. tropicalis) muscle lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 51 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-gamma Tubulin antibody [EPR16793] (ab179503) at 1/20000 dilution

Lane 1 : UMNSAH/DF-1 (Transformed chicken embyronic fibroblast cells) whole cell lysates
Lane 2 : BHK (Hamster kidney fibroblast cells) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 51 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-gamma Tubulin antibody [EPR16793] (ab179503) at 1/2000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Mouse spleen lysates
Lane 3 : Rat brain lysates
Lane 4 : Rat heart lysates
Lane 5 : Rat kidney lysates
Lane 6 : C6 (Rat glial tumor cells) whole cell lysates
Lane 7 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates
Lane 8 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 9 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

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: 51 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling gamma Tubulin with ab179503 at 1/250 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasmic staining on epithelial cells of mouse testis is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

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

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling gamma Tubulin with ab179503 at 1/250 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasmic staining on tumor cells of breast carcinoma is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

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

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling gamma Tubulin with ab179503 at 1/250 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm staining on epithelial cells of rat testis is observed. Counter stained with Hematoxylin.

 

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

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

Flow cytometric analysis of 2% paraformaldehyde-fixed UMNSAH/DF-1 (Transformed chicken embyronic fibroblast cells) cells labeling gamma Tubulin with ab179503 at 1/80 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an 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.

Gamma Tubulin was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab179503 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab179503 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution. Lane 1: HeLa whole cell extract. Lane 2: PBS instead of HeLa whole cell extract.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.