Anti-Eg5 antibody [mAbcam 51976]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Eg5 antibody [mAbcam 51976] (AB51976)

ab51976 staining Eg5 in HeLa 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 ab51976 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min).Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IP

Unknown

Immunoprecipitation - Anti-Eg5 antibody [mAbcam 51976] (AB51976)

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

All lanes:

Immunoprecipitation - Anti-Eg5 antibody [mAbcam 51976] (ab51976)

Predicted band size: 119 kDa

false

• WB

Project

Western blot - Anti-Eg5 antibody [mAbcam 51976] (AB51976)

All lanes:

Western blot - Anti-Eg5 antibody [mAbcam 51976] (ab51976) at 5 µg/mL

Lane 1:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Lane 2:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 119 kDa

Observed band size: 120 kDa

false