All lanes : Anti-SSB antibody [mAbcam75927] (ab75927) at 5 µg/ml

Lane 1 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 3 : Raji (Human Burkitt's lymphoma cell line) Nuclear Lysate - tumor cell line (ab30127)
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) Nuclear Lysate
Lane 5 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 6 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lane 7 : Heart (Human) Whole Cell Lysate - fetal normal tissue

Lysates/proteins at 20 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.

Predicted band size: 47 kDa
Observed band size: 47 kDa


Exposure time: 30 seconds

IHC image of SSB staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab75927, 0.5µg/ml overnight at +4°C. An HRP-conjugated secondary (Ab97245, 1/200 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

SSB was immunoprecipitated using 0.5mg Hek293 whole cell extract, 5µg of Mouse monoclonal to SSB 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, Hek293 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°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab75927.

Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

Band: 47kDa; SSB

ICC/IF image of ab75927 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab75927 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse IgG (H+L)(ab96879) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in Hek293, HepG2, and MCF-7 cell types Methanol fixed (100%, 5 min) at 5ug/ml

Anti-SSB antibody [mAbcam75927] (ab75927) at 1 µg/ml + Recombinant Human SSB protein (ab84477) at 0.1 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: 47 kDa


Exposure time: 30 seconds

Overlay histogram showing Ramos cells stained with ab75927 (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 (ab75927, 0.5µg/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 IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Ramos cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

IHC image of SSB staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab75927, 5µg/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.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.