Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling alpha Tubulin with ab52866 at 1/500 dilution. The cells were permeabilised with 0.1% Triton X-100. Anti-rabbit Alexa Fluor® 488 (ab150077) at 1/400 dilution was used as the secondary antibody (green). The confocal image shows microtubules staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 and anti-mouse AlexaFluor® 594 (ab150120) at 1/500 dilution (red).
The negative controls are as follows:
1. ab52866 at 1/500 dilution followed by anti-mouse AlexaFluor® 594 (ab150120) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by anti-rabbit Alexa Fluor® 488 (ab150077) at 1/400 dilution.

 

All lanes : Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) at 1/10000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) Whole cell lysates
Lane 2 : PC-12 (Rat adrenal gland heochromocytoma) whole cell lysates
Lane 3 : NIH/3T3( Mouse embryonic fibroblast)whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 50 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling alpha Tubulin with ab52866 at   followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on human stomach.The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol  permeabilized HepG2 (Human hepatocellular carcinoma epithelial cell) cells labelling alpha Tubulin with ab52866 at 1/2000 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.  

All lanes : Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) at 1/5000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : C6 (Rat glial tumor cell line) whole cell lysates
Lane 3 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysates
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysates
Lane 5 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell 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: 50 kDa

Immunohistochemistry analysis of paraffin-embedded Pig kidney tissue labeling alpha Tubulin with ab52866 at a 1/1000 dilution. Cytoplasmic staining on Pig kidney tubule and weak on glomerulus shown. Anti-Rabbit HRP (ab97051) used at a 1/100 dilution. Counter stained with Hematoxylin.

Inset image: negative control obtained using PBS instead of ab52866, secondary antibody is Anti-Rabbit HRP (ab97051) at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Flow cytometry analysis of 2% paraformaldehyde fixed HepG2 (human liver hepatocellular carcinoma cell line) cells labeling alpha Tubulin with ab52866 at 1/130 dilution (red line). Secondary antibody used is a goat anti rabbit IgG (FITC) at 1/150 dilution. The isotype control is rabbit monoclonal IgG (black line). The unlabeled control is cells without incubation with primary and secondary antibodies (blue line).

Immunohistochemistry analysis of paraffin-embedded Rat kidney tissue labeling alpha Tubulin with ab52866 at a 1/1000 dilution. Cytoplasmic staining on Rat kidney tubule and weak on glomerulus shown. Secondary antibody Anti-Rabbit HRP (ab97051) used at a 1/500 dilution. Counter stained with Hematoxylin.

Inset image: negative control obtained using PBS instead of ab52866, secondary antibody is Anti-Rabbit HRP (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

AJAP1 co-localizes with microtubules in HUVECs

The association of AJAP1 with microtubules in HUVECs is lost upon microtubule destruction. Treatment with 12.5 µM nocodazole for 24 h shows destruction of the microtubule network and loss of AJAP1 tubular localization. For a negative control, HUVECs are treated with DMSO for 24 h. Cell nuclei were counterstained with DAPI (cyan). Microscope: Zeiss LSM 780; objective lens: 63×/1.40 oil; scale bar: 25 µm.

Incubated overnight at 4°C with ab52866.

(From Figure 3E of Hotte et al)

All lanes : Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) at 1/20000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 4 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate

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: 50 kDa

Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) at 1/50000 dilution + Rat brain lysates at 10 µg

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

Predicted band size: 50 kDa

ab52866 staining alpha Tubulin in 293 Human embryonic kidney cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 10% serum for 2 hours at 23°C. Samples were incubated with primary antibody (1/200 in 0.5% saponin) for 2 hours at 23°C. An Alexa Fluor^®555-conjugated Goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody. Nuclei were counterstained with DAPI.

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Immunohistochemistry analysis of paraffin-embedded Mouse kidney tissue labeling alpha Tubulin with ab52866 at a 1/1000 dilution. Cytoplasmic staining on Mouse kidney tubule shown. Secondary antibody Anti-Rabbit HRP (ab97051) used at a 1/500 dilution. Counter stained with Hematoxylin.

Inset image: negative control obtained using PBS instead of ab52866, secondary antibody is Anti-Rabbit HRP (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry analysis of paraffin-embedded Human breast cancer labeling alpha Tubulin with ab52866 at a 1/1000 dilution. Cytoplasmic staining on cancer cells shown. Secondary antibody ab97051 Goat Anti-Rabbit IgG H&L (HRP) used at a 1/500 dilution. Counter stained with Hematoxylin.

Inset image: negative control obtained using PBS instead of ab52866, secondary antibody is Anti-Rabbit HRP (ab97051) at 1/500 dilution.

 

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) at 1/50000 dilution + Human fetal kidney lysates at 10 µg

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

Predicted band size: 50 kDa

All lanes : Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 4 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
Lane 5 : NIH/3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 6 : PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) at 1/10000 dilution

Predicted band size: 50 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab52866 overnight at 4°C. Antibody binding was detected using Anti-Rabbit Alexa Fluor® 790 (ab175781) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) at 1/5000 dilution + Pig skeletal muscle lysates at 20 µg

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

Predicted band size: 50 kDa