Anti-SIRT6 抗体 [EPR26255-85] (ab289970)

Blocking buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.
Diluting buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBST.

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

False colour image of Western blot: Anti-SIRT6 antibody [EPR26255-85](ab289970) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab289970 was shown to bind specifically to SIRT6. Two bands were observed at 43/36 kDa in wild-type SIRT6 cell lysates with no signal observed at this size in SIRT6 knockout cell line ab265054 (knockout cell lysate ab257673). To generate this image, wild-type and SIRT6 knockout HeLa cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/10000 dilution.

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

Exposure times: Lane 1: 26 seconds Lane 2-4: 7.75 seconds

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling SIRT6 with ab289970 at 1/2000 followed by LeicaDS9800 (Bond™, Polymer Refine Detection). Positive staining on human colon (PMID 30333863) (PMID 29387864). The section was incubated with ab289970 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is LeicaDS9800 (Bond™, Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Parental HeLa cell pellet labelling SIRT6 with ab289970 at 1/10000  followed by LeicaDS9800 (Bond™, Polymer Refine Detection) was used. Positive staining on Parental HeLa cell pellet (A), no staining on SIRT6 KO HeLa cell pellet (B). The section was incubated with ab289970 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is LeicaDS9800 (Bond™, Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human thymus tissue labelling SIRT6 with ab289970 at 1/2000 followed by LeicaDS9800 (Bond™, Polymer Refine Detection). Positive staining on human thymus (PMID 29387864). The section was incubated with ab289970 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is LeicaDS9800 (Bond™, Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

ab289970 staining SIRT6 in wild-type HeLa (human cervix adenocarcinoma epithelial cell line) cells and SIRT6 knockout HeLa (human cervix adenocarcinoma epithelial cell line) cells (ab265054). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then incubated with ab289970 at 1/1000 dilution and ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibody at 1/1000 dilution. Nuclear DNA was labelled in blue with DAPI. Confocal image showing nuclear staining in parental Hela cell line and no staining in SIRT6 KO Hela cell line.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Parental HeLa (Human cervix adenocarcinoma epithelial cell, Right) / SIRT6 KO HeLa (Left) cells labelling SIRT6 with ab289970 at 1/5000 dilution (0.01µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Positive staining on HeLa cells (ab255448), while no staining on SIRT6 knockout HeLa cells (ab265054).

SIRT6 was immunoprecipitated from HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab289970 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab289970 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg

Lane 2: ab289970 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab289970 in HeLa whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 7.75 seconds