Anti-BST2/Tetherin antibody [EPR20202-150]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-BST2/Tetherin antibody [EPR20202-150] (AB243230)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling BST2/Tetherin with ab243230 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody (Green). Confocal image showing cytoplasmic staining in HeLa cells (PMID : 20529266).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-BST2/Tetherin antibody [EPR20202-150] (AB243230)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-937 (human histiocytic lymphoma cell line) cells labeling BST2/Tetherin with ab243230 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody (Green). Confocal image showing cytoplasmic staining in U-937 cells (PMID : 20529266).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt

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Flow Cytometry - Anti-BST2/Tetherin antibody [EPR20202-150] (AB243230)

Flow cytometric analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling BST2/Tetherin with ab243230 at 1/700 dilution (Red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

• IP

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Immunoprecipitation - Anti-BST2/Tetherin antibody [EPR20202-150] (AB243230)

BST2/Tetherin was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab243230 at 1/40 dilution. Western blot was perfromed on the immunoprecipitate using ab243230 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used afor detection at 1/5000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (input)
Lane 2 : ab243230 IP in HeLa whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab243230 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST
Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-BST2/Tetherin antibody [EPR20202-150] (ab243230)

Predicted band size: 20 kDa

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• WB

Lab

Western blot - Anti-BST2/Tetherin antibody [EPR20202-150] (AB243230)

Lanes 1 - 4 : Merged signal (red and green). Green - ab243230 observed at 30 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab243230 was shown to react with BST2/Tetherin in wild-type HeLa cells in Western blot with loss of signal observed in BST2 knockout samples. Wild-type HeLa and BST2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab243230 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-BST2/Tetherin antibody [EPR20202-150] (ab243230) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Bst2 knockout HeLa cell lysate at 20 µg

Lane 3:

Wild-type A431 cell lysate at 20 µg

Lane 4:

BST2 knockout A431 cell lysate at 20 µg

Predicted band size: 20 kDa

Observed band size: 30 kDa

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• WB

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Western blot - Anti-BST2/Tetherin antibody [EPR20202-150] (AB243230)

Blocking/Diluting buffer and concentration : 5% NFDM/TBST

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 19196977; PMID : 19737401).

BST2/Tetherin is a glycosylated protein, its calculated MW is 20 kDa, and the observed MW is 35 kDa, which is consistent with the literature.

Both 35 and 70 kDa bands were detected under the reducing condition, whereas under the non-reducing condition, only the 70 kDa band was detected.

All lanes:

Western blot - Anti-BST2/Tetherin antibody [EPR20202-150] (ab243230) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate in the loading buffer containing DTT at 20 µg

Lane 2:

HeLa whole cell lysate in the loading buffer without DTT at 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate in the loading buffer containing at 20 µg

Lane 4:

K562 whole cell lysate in the loading buffer without DTT at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 20 kDa

Observed band size: 35 kDa,70 kDa

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Exposure time: 48s

• WB

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Western blot - Anti-BST2/Tetherin antibody [EPR20202-150] (AB243230)

Blocking/Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-BST2/Tetherin antibody [EPR20202-150] (ab243230) at 1/1000 dilution

All lanes:

U-937 (human histiocytic lymphoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 20 kDa

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Exposure time: 125s