Anti-SSB antibody [mAbcam75927]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SSB antibody [mAbcam75927] (AB75927)

ab75927 staining SSB in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 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 ab75927 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SSB antibody [mAbcam75927] (AB75927)

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 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 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-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SSB antibody [mAbcam75927] (AB75927)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SSB antibody [mAbcam75927] (AB75927)

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.

• IP

Lab

Immunoprecipitation - Anti-SSB antibody [mAbcam75927] (AB75927)

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

All lanes:

Immunoprecipitation - Anti-SSB antibody [mAbcam75927] (ab75927)

Predicted band size: 46 kDa

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Exposure time: 20min

• WB

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Western blot - Anti-SSB antibody [mAbcam75927] (AB75927)

All lanes:

Western blot - Anti-SSB antibody [mAbcam75927] (ab75927) at 5 µg/mL

Lane 1:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg

Lane 2:

HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 20 µg

Lane 3:

Raji (Human Burkitt's lymphoma cell line) Nuclear Lysate - tumor cell line (<a href='/en-us/products/unavailable/raji-human-burkitt-s-lymphoma-cell-line-nuclear-lysate-tumor-cell-line-ab30127'>ab30127</a>) at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) Nuclear Lysate at 20 µg

Lane 5:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 20 µg

Lane 6:

Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 20 µg

Lane 7:

Heart (Human) Whole Cell Lysate - fetal normal tissue at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 46 kDa

Observed band size: 47 kDa

true

Exposure time: 30s

• WB

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Western blot - Anti-SSB antibody [mAbcam75927] (AB75927)

All lanes:

Western blot - Anti-SSB antibody [mAbcam75927] (ab75927) at 1 µg/mL

All lanes:

Western blot - Recombinant Human SSB protein (<a href='/en-us/products/proteins-peptides/recombinant-human-ssb-protein-ab84477'>ab84477</a>) at 0.1 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 46 kDa

true

Exposure time: 30s

• WB

Unknown

Western blot - Anti-SSB antibody [mAbcam75927] (AB75927)

All lanes:

Western blot - Anti-SSB antibody [mAbcam75927] (ab75927) at 1 µg/mL

All lanes:

Western blot - Recombinant Human SSB protein (<a href='/en-us/products/proteins-peptides/recombinant-human-ssb-protein-ab84477'>ab84477</a>) at 0.1 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 46 kDa

true

Exposure time: 30s

• WB

CiteAb

Western blot - Anti-SSB antibody [mAbcam75927] (AB75927)

SSB western blot using anti-SSB antibody [mAbcam75927] ab75927. Publication image and figure legend from Spurlock, C. F., Tossberg, J. T., et al., 2015, Genome Biol, PubMed 25885816.

ab75927 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab75927 please see the product overview.

Ro60 and La proteins are depressed in RRMS. (A) Ro60 (TROVE2) and La (SSB) transcript levels in CTRL (N = 24), CIS-MS (N = 16), RRMS (N = 22), RA (N = 18), SLE (N = 24), NMO (N = 22), and PD (N = 19) were determined by quantitative PCR after cDNA synthesis using oligo-dT. Results are normalized to CTRL = 1.0 after normalization to transcript levels of GAPDH, error bars are S.D. (B) As in (A) using whole genome RNA-sequencing data. (C) Western blot to determine Ro60 and La protein levels in PBMC from CTRL (N = 9) and RRMS (N = 8). (D) Quantitative estimates of protein abundance relative to β-actin. *p < 0.05, **p < 0.01.

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