Anti-beta Tubulin antibody - Loading Control (ab6046)

Western blot

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 beta Tubulin peptide (ab20775) at 1 µg/ml
Lane 6 : A431 Cell lysate with beta Tubulin peptide (ab20775) at 1 µg/ml
Lane 7 : MCF7 Cell lysate with beta Tubulin peptide (ab20775) at 1 µg/ml
Lane 8 : 293 Cell lysate with beta Tubulin peptide (ab20775) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 50 kDa
Observed band size : 55 kDa (why is the actual band size different from the predicted?)


Exposure time : 10 seconds

Immunoprecipitation - Anti-beta Tubulin antibody - Loading Control (ab6046)

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.

Immunocytochemistry/ Immunofluorescence - beta Tubulin antibody - Loading Control (ab6046)

Immunofluorescent imaging of human cells (U2OS) with ab6046 confirms the specificity of this anti-tubulin antibody, with the expected fine filamentous and reticular cytoplasmic distribution in these confluent U2OS cells. Note the complete absence of background nuclear staining. 

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 for 30 minutes.  All blocking and incubation steps carried out at 37 degrees C. Nuclei were stained with Hoechst stain.

Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK

Immunofluorescence - beta Tubulin antibody - Loading Control (ab6046)

This image shows a HEK 293 cell expressing rhodopsin
(green) and tubulin (red), DAPI staining (blue) for nucleus.

Immunocytochemistry/ Immunofluorescence - beta Tubulin antibody - Loading Control (ab6046)

ICC/IF image of ab6046 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab6046, 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-rabbit 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - beta Tubulin antibody - Loading Control (ab6046)

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.

Immunohistochemistry (Frozen sections) - beta Tubulin antibody - Loading Control (ab6046)

ab6046 staining beta Tubulin in human stomach tissue by Immunohistochemistry (frozen sections). Tissue was fixed with acetone and then blocked with 5% serum for 1 hour at 23ºC followed by incubation with the primary antibody at a 1/200 dilution for 1 hour at 23ºC. An undiluted HRP-conjugated goat polyclonal was used as secondary antibody.

Image courtesy of an anonymous Abreview.

Immunocytochemistry/ Immunofluorescence - beta Tubulin antibody - Loading Control (ab6046)

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 +4ºC. 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.