Merged signal (red and green). Green - ab7291 observed at52 kDa. Red - loading control, ab181602, observed at 38 kDa.

All samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. ab7291 and ab181602 (Rabbit antiGAPDH loading control) were incubated overnight at 4°C at1/1,000and 1/20,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbedab216772and Goat anti-Rabbit IgG H&L (IRDye^®680RD) preabsorbedab216777secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab7291 staining alpha tubulin in human breast cancer cell line by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS and blocked with 2% bovine serum albumin in sodium phosphate buffer. Cells were co-stained with anti-pericentrin using ab4448 at 1:500 dilution and ab7291 at 1:500 dilution. Alexa Fluor^®633 goat anti mouse and Alexa Fluor^® 488 goat anti-rabbit (1:500 dilution) was used as secondary antibodies. DAPI was used as a nuclei counterstain. Representative images of mitotic cells with bipolar or multipolar spindles.

ab7291 staining alpha Tubulin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab7291 at a working concentration of 0.5μg/ml and ab190573, Rabbit monoclonal [EP1332Y] to alpha Tubulin(Alexa Fluor^® 647, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor^® 488 (ab150117) at 2 μg/ml (shown in green).Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal in 100% methanol (5 min) fixed SV40 cells under the same testing conditions.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

IHC image of ab7291 staining alpha Tubulin in human colon formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7291, 5ug/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Overlay histogram showing HeLa cells stained with ab7291 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab7291, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was an anti-mouse DyLight^® 488 (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

ab7291 stainingalpha-Tubulin inCaco-2 cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab7291 at 1μg/ml and ab6046 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor^® 488 (ab150117) at 2 μg/ml (shown in green) and anti-rabbit Alexa Fluor^® 594 (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Negative controls:1 – Rabbit primary antibody and anti-mouse secondary antibody.2 – Mouse primary antibody and anti-rabbit secondary antibody.Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

ab7291 stainingalpha TubulininNIH3T3 cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab7291 at 1μl/ml and ab6046 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse Alexa Fluor^® 488 (ab150117) at 2 μg/ml (shown in green) and anti-rabbit Alexa Fluor^® 594 (ab150088) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Negative controls:1 – Rabbit primary antibodyand anti-mouse secondary antibody.2 – Mouse primary antibody and anti-rabbit secondary antibody.Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

See Abreview

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 2% Bovine Serum Albumin before being incubated with ab7291 overnight at 4°C. Antibody binding was detected using an anti-mouse HRP (ab97040), and visualised using ECL development solution ab133406

FABP4 (green) was detected using FABP4 primary antibody (ab92501; diluted 1/1000). Alpha tubulin (red) was detected usingthe mouse monoclonal (ab7291) antibody. Cells were imaged by confocal microscopy, using z-stack for adipocyte-like cells.

IHC image of ab7291 staining alpha Tubulin in rat colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab7291, 0.5ug/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

ICC/IF image of ab7291 stained human HeLa cells. The cells were methanol fixed (5 min)and incubated with the antibody (ab7291, 1µg/ml) for 1h at room temperature. The secondary antibody (green)was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor^® 594 WGA was used to label plasma membranes (red).

Immunofluorescent imaging of human cells (U2OS) with ab7291 reveals a delicate network of alpha-tubulin (green) located exclusively in the cytoplasm. The nucleus is stained blue.

IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes. All blocking and incubation steps carried out at 37 degrees.