All lanes : Anti-TTK/Mps1 antibody [N1] - BSA free (ab11108) at 1 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 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 4 : Jurkat nuclear extract lysate (ab14844)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 95 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?


Exposure time: 8 minutes


NOTE: MILK was used to block non-specific binding, rather than BSA.

TTK/Mps1 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Mouse monoclonal to Mps1 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, Jurkat 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 ab11108.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: 100kDa: TTK/Mps1 

ICC/IF image of ab11108 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab11108, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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).

Overlay histogram showing HeLa cells stained with ab11108 (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 (ab11108, 2µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (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.

Anti-TTK/Mps1 antibody [N1] - BSA free (ab11108) at 10 µg/ml + Recombinant human TTK/Mps1 protein (ab89589) at 0.01 µg

Secondary
Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 95 kDa


Exposure time: 8 minutes

ab11108 recognizes the full length tagged recombinant TTK/Mps1 protein (ab89589) which has an expected molecular weight of 130 kDa.