ICC/IF image of ab22758 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab22758, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

IHC image of Nucleolin staining in human tonsil FFPE section, performed on a 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 ab22758, 1µg/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.

All lanes : Anti-Nucleolin antibody (ab22758) at 1 µg/ml

Lane 1 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 2 : Brain (Mouse) Tissue Lysate
Lane 3 : Pancreas (Mouse) Tissue Lysate
Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 76 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?
Additional bands at: 60 kDa. We are unsure as to the identity of these extra bands.

Nucleolin was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to Nucleolin 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 ab22758.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 100kDa: Nucleolin.

Lane 1 : Marker
Lanes 2-5 : Anti-Nucleolin antibody (ab22758) at 1 µg/ml

Lane 2 : Jurkat whole cell lysate (ab7899) at 20 µg
Lane 3 : A-431 whole cell lysate (ab7909) at 20 µg
Lane 4 : Jurkat whole cell lysate (ab7899) at 20 µg with Human Nucleolin peptide (ab25315) at 1 µg/ml
Lane 5 : A-431 whole cell lysate (ab7909) at 20 µg with Human Nucleolin peptide (ab25315) at 1 µg/ml

Secondary
Lanes 2-5 : Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 dilution


Performed under reducing conditions.

Predicted band size: 76 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

ab22758 staining nucleolin in HeLa cells treated with Triptolide from Tripterygium wilfordii (ab120720), by ICC/IF. Changes in nuclear localization of nucleolin (from nuclelous to whole nucleous) correlates with increased concentration of Triptolide from Tripterygium wilfordii, as described in literature.
The cells were incubated at 37°C for 1h in media containing different concentrations of ab120720 (Triptolide from Tripterygium wilfordii ) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab22758 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody.