ab6161 staining Tubulin in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab6161 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150165, Goat polyclonal Secondary Antibody to Rat IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Western blot against tubulin with ab6161 at 1/3000.  Secondary Rabbit anti-Rat IgG HRP (ab6734)was used at 1/2000.  Exposure time: 2mins.

Lane 1: 20µg/lane HeLa (Human) whole cell lysates (ab7898).

Lane 2: 20µg/lane 3T3 (Mouse) whole cell lysate (ab7901).

Lane 3: 20µg/lane Rat brain tissue lysate (ab7942).

Confocal image of  21 day in vitro rat hippocampal neurons, stained with rat monoclonal antibody to Tubulin - Microtubule Marker (ab6161) in green at 1/500 and Microtubule Associated protein 2 in blue.

This picture was kindly supplied as part of the review submitted by Dr Jonathon Burman.

Overlay histogram showing HeLa cells stained with ab6161 (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 (ab6161, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rat IgG2a [aRTK2758] (ab18450, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Cultured human macrophages were used with ab6161 at 1/1000 for immunofluorescence. Cells were fixed with cold 2% formaldehyde for 20mins.

Green staining is Alexa 568, Blue staining is DAPI stain.

This cell represents a young macrophage, the staining patterns varied as the cells aged in culture.

All lanes : Anti-Tubulin antibody [YOL1/34] - Microtubule Marker (ab6161) at 1/2000 dilution

All lanes : Yeast (Saccharomyces cerevisiae) whole cell extract prepared by bead-beating

Lysates/proteins at 5 µg per lane.

Secondary
All lanes : HRP conjugated goat anti-rat antibody

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 30 seconds

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ICC/IF image of ab6161 stained human HepG2 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab6161, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rat IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa, HEK 293 and MCF7 cells.

Anti-Tubulin antibody [YOL1/34] - Microtubule Marker (ab6161) at 1 µg/ml + Brain (Rat) Tissue Lysate at 10 µg

Secondary
Rabbit polyclonal to Rat IgG - H&L (HRP) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 54 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

ab6161 staining mouse NIH 3T3 fibroblast cells by ICC/IF. Cells were PFA fixed  and permeabilized in 0.2% Triton X-100 prior to blocking in 5% BSA for 45 minutes at RT.  The primary antibody was diluted 1/1000 and incubated with the sample for 1 hour.  An Alexa Fluor^® 568 conjugated goat anti-rat antibody, diluted 1/3000, was used as the secondary.

 

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ab6161 staining tubulin HeLa cells treated with anisomycin (ab120495), by ICC/IF. Increase in tubulin expression correlates with increased concentration of anisomycin as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab120495 (anisomycin) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab6161 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rat polyclonal antibody (ab98386) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.