ICC/IF image of ab51976 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab51976, 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).

All lanes : Anti-Eg5 antibody [mAbcam 51976] (ab51976) at 5 µg/ml

Lane 1 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Rabbit polyclonal to Mouse IgG - H&L (HRP) at 1/2000 dilution

Performed under reducing conditions.

Predicted band size: 120 kDa
Observed band size: 120 kDa

Eg5 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Mouse monoclonal to Eg5 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 ab51976.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: 120kDa: Eg5