All lanes : Anti-Lamin B2 antibody [LN43] (ab8983) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate
Lane 4 : HL60 (Human promyelocytic leukemia cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 75 kDa why is the actual band size different from the predicted?
Additional bands at: 37 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

ab8983 immunohistochemistry on a frozen section of human kidney showing nuclear lamina staining in the ductal epithelium.

IHC-Fr of human kidney showing nuclear lamina staining in the ductal epithelium.  

Lamin B2 - Nuclear Envelope Marker was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to Lamin B2 - Nuclear Envelope Marker (ab8983) 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 ab8983.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: 73kDa: Lamin B2

Overlay histogram showing HeLA cells stained with ab8983 (red line). The cells were fixed with 100% 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 (ab7481) / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab8983, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.

IF staining of a 9 days old zebrafish embryo.