All lanes : Anti-beta Tubulin antibody - Loading Control (ab6046) at 1/500 dilution

Lane 1 : HeLa Cell lysate
Lane 2 : A431 Cell lysate
Lane 3 : MCF7 Cell lysate
Lane 4 : 293 Cell lysate
Lane 5 : HeLa Cell lysate with Human beta Tubulin peptide (ab20775) at 1 µg/ml
Lane 6 : A431 Cell lysate with Human beta Tubulin peptide (ab20775) at 1 µg/ml
Lane 7 : MCF7 Cell lysate with Human beta Tubulin peptide (ab20775) at 1 µg/ml
Lane 8 : 293 Cell lysate with Human beta Tubulin peptide (ab20775) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 50 kDa


Exposure time: 10 seconds

ICC/IF image of ab6046 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6046, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab150081 Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

The negative control (inset) is a secondary-only assay to demonstrate low non-specific binding of the secondary antibody.

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

ICC/IF image of ab6046 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6046, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Beta Tubulin was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Tubulin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab6046.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 50kDa: beta Tubulin.

IHC image of beta Tubulin staining in human liver carcinoma FFPE section, performed on a Leica Bond^TM system using the standard protocol F. 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 ab6046, 5µg/ml, 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.