ab11315 staining Beta IV tubulin in MCF7 cells. The cells were fixed with 100% methanol (5 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 ab11315 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) 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.

All lanes : Anti-beta IV Tubulin antibody [ONS.1A6] (ab11315) at 5 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 4 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate
Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 6 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 50 kDa
Additional bands at: 52 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

This blot was produced using a 10% 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 3% Milk before being incubated with ab11315 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.

IHC image of beta IV Tubulin staining in Human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab11315, 1µg/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.

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.

Immunofluorescence analysis of MCF-7 Cells (human breast cancer cell line), staining beta IV Tubulin with ab11315.

Cells on coverslip were fixed with -20°C Methanol for 5 min before permeabilization with 0.2% Triton X-100 in PBS for 10 min. Cells were incubated with the primary antibody with a dilution 1/50 for 1 hour. After brief washing with PBS, cells were incubated with TRITC conjugated secondary antibody with a dilution 1/50 for 45 min and washed with PBS to be examined under fluorescence microscope.

Immunofluorescence analysis of MCF-7 Cells (human breast cancer cell line), staining beta IV Tubulin with ab11315.

Cells on coverslip were fixed with -20°C Methanol for 5 min before permeabilization with 0.2% Triton X-100 in PBS for 10 min. Cells were incubated with the primary antibody with a dilution 1/50 for 1 hour. After brief washing with PBS, cells were incubated with TRITC conjugated secondary antibody with a dilution 1/50 for 45 min and washed with PBS to be examined under fluorescence microscope.