BST2/Tetherin was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab243229 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab243229 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 µg (input).
Lane 2: ab243229 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab243229 in HeLa whole cell lysate.

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

Flow cytometric analysis of U-937 (human histiocytic lymphoma cell line) cell line labeling BST2/Tetherin with ab243229 at 1/500 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) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

Flow cytometric analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line labeling BST2/Tetherin with ab243229 at 1/500 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) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-937 (human histiocytic lymphoma cell line) cells labeling BST2/Tetherin with ab243229 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (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.

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 ab243229 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (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.

Anti-BST2/Tetherin antibody [EPR20202-169] (ab243229) at 1/1000 dilution + U937 (human histiocytic lymphoma cell line) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 20 kDa
Observed band size: 35,70 kDa why is the actual band size different from the predicted?

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: 19737401)

All lanes : Anti-BST2/Tetherin antibody [EPR20202-169] (ab243229) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinom), whole cell lysate, in the loading buffer containing DTT
Lane 2 : HeLa whole cell lysate in the loading buffer without DTT
Lane 3 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate, in the loading buffer containing DTT
Lane 4 : K562 whole cell lysate in the loading buffer without DTT

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 20 kDa
Observed band size: 35,70 kDa why is the actual band size different from the predicted?


Exposure time: 48 seconds

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 20kDa, and the observed MW is 35 kDa, which is consistent to 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.