Anti-Selenium Binding Protein 1/SBP antibody

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Selenium Binding Protein 1/SBP antibody (AB90135)

IHC image of ab90135 staining in Normal Human Liver formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab90135, 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Selenium Binding Protein 1/SBP antibody (AB90135)

ICC/IF image of ab90135 stained MCF-7 cells. The cells were 4% PFA fixed (10 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 (ab90135, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) 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 4% PFA fixed (10 min) HeLa cells at 5µg/ml, and in 100% Methanol fixed (5 min) HeLa, and Hek293 cells at 5µg/ml.

• IP

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Immunoprecipitation - Anti-Selenium Binding Protein 1/SBP antibody (AB90135)

Selenium Binding Protein 1/SBP was immunoprecipitated using 0.5mg Mouse Liver tissue lysate, 5μg of Rabbit polyclonal to Selenium Binding Protein 1/SBP 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, Mouse Liver tissue lysate 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 ab90135.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 52kDa; Selenium Binding Protein 1/SBP

All lanes:

Immunoprecipitation - Anti-Selenium Binding Protein 1/SBP antibody (ab90135)

Predicted band size: 52 kDa

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• WB

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Western blot - Anti-Selenium Binding Protein 1/SBP antibody (AB90135)

Lanes 1-3 : Merged signal (red and green). Green - ab90135 observed at 60 kDa. Red - loading control ab8245 observed at 36 kDa.

ab90135 Anti-Selenium Binding Protein 1/SBP antibody was shown to specifically react with Selenium Binding Protein 1/SBP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265279 (knockout cell lysate ab257662) was used. Wild-type and Selenium Binding Protein 1/SBP knockout samples were subjected to SDS-PAGE. ab90135 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Selenium Binding Protein 1/SBP antibody (ab90135) at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

SELENBP1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SELENBP1 (Selenium Binding Protein 1/SBP) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-selenbp1-selenium-binding-protein-1-sbp-knockout-hela-cell-line-ab265279'>ab265279</a>)

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

SW 480 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lane 4:

<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a> at 1/10000 dilution

Predicted band size: 52 kDa

Observed band size: 60 kDa

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• WB

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Western blot - Anti-Selenium Binding Protein 1/SBP antibody (AB90135)

All lanes:

Western blot - Anti-Selenium Binding Protein 1/SBP antibody (ab90135) at 1 µg/mL

Lane 1:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg

Lane 2:

Human colon tissue lysate - total protein (<a href='/en-us/products/unavailable/human-colon-tissue-lysate-total-protein-ab30051'>ab30051</a>) at 10 µg

Lane 3:

Human spleen tissue lysate - total protein (<a href='/en-us/products/unavailable/human-spleen-tissue-lysate-total-protein-ab29699'>ab29699</a>) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 52 kDa

Observed band size: 50 kDa,52 kDa

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Exposure time: 1min

• WB

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Western blot - Anti-Selenium Binding Protein 1/SBP antibody (AB90135)

All lanes:

Western blot - Anti-Selenium Binding Protein 1/SBP antibody (ab90135) at 1 µg/mL

Lane 1:

Liver (Mouse) Tissue Lysate at 10 µg

Lane 2:

Lung (Mouse) Tissue Lysate at 10 µg

Lane 3:

Liver (Rat) Tissue Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

true

Exposure time: 1min