Anti-SIRT6 antibody [EPR18463]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SIRT6 antibody [EPR18463] (AB191385)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (Human colorectal carcinoma cell line) cells labeling SIRT6 with ab191385 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HCT 116 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab191385 at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SIRT6 antibody [EPR18463] (AB191385)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling SIRT6 with ab191385 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab191385 at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.

-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-SIRT6 antibody [EPR18463] (AB191385)

SIRT6 was immunoprecipitated from 1 mg of Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate with ab191385 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab191385 at 1/2000 dilution.

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : Jurkat whole cell lysate 10μg (Input).

Lane 2 : ab191385 IP in Jurkat whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) IP instead of ab191385 in Jurkat whole cell lysate.

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

Exposure time : 3 seconds.

The observed MW and doublets are consistent with what has been described in the literature. Two bands run closely together as doublets representing distinct isoforms; see UniProt annotation and PMID 24169447.

All lanes:

Immunoprecipitation - Anti-SIRT6 antibody [EPR18463] (ab191385)

Predicted band size: 39 kDa

Observed band size: 42 kDa

false

• WB

Lab

Western blot - Anti-SIRT6 antibody [EPR18463] (AB191385)

Lanes 1-2 : Merged signal (red and green). Green - ab191385 observed at 40 kDa. Red - loading control ab7291 observed at 50 kDa.

ab191385 Anti-SIRT6 antibody [EPR18463] was shown to specifically react with SIRT6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265054 (knockout cell lysate ab257673) was used. Wild-type and SIRT6 knockout samples were subjected to SDS-PAGE. ab191385 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 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-SIRT6 antibody [EPR18463] (ab191385) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

SIRT6 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SIRT6 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-sirt6-knockout-hela-cell-line-ab265054'>ab265054</a>)

Predicted band size: 39 kDa

Observed band size: 40 kDa,42 kDa

false

• WB

Supplier Data

Western blot - Anti-SIRT6 antibody [EPR18463] (AB191385)

Blocking/Dilution buffer : 5% NFDM/TBST.

SIRT6 is detected in nuclear fractions.

All lanes:

Western blot - Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/5000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cytoplasmic lysate at 10 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) nuclear lysate at 10 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: 39 kDa

Observed band size: 42 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-SIRT6 antibody [EPR18463] (AB191385)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed MW and doublets are consistent with what has been described in the literature. Two bands run closely together as doublets representing distinct isoforms; see UniProt annotation and PMID 24169447.

All lanes:

Western blot - Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/10000 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 1/50000 dilution

Predicted band size: 39 kDa

Observed band size: 42 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-SIRT6 antibody [EPR18463] (AB191385)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed MW and doublets are consistent with what has been described in the literature. Two bands run closely together as doublets representing distinct isoforms; see UniProt annotation and PMID 24169447.

All lanes:

Western blot - Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/10000 dilution

All lanes:

Jurkat (Human T cell leukemia cell line from peripheral blood) 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 1/50000 dilution

Predicted band size: 39 kDa

Observed band size: 42 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-SIRT6 antibody [EPR18463] (AB191385)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed MW and doublets are consistent with what has been described in the literature. Two bands run closely together as doublets representing distinct isoforms; see UniProt annotation and PMID 24169447.

All lanes:

Western blot - Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/10000 dilution

All lanes:

NIH/3T3 (Mouse embryonic fibroblast cell line) 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 1/50000 dilution

Predicted band size: 39 kDa

Observed band size: 39 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-SIRT6 antibody [EPR18463] (AB191385)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed MW is consistent with what has been described in the literature (PMID 24169447).

All lanes:

Western blot - Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/2000 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

Rat spleen lysate at 10 µg

Lane 3:

C6 (Rat glial tumor cell line) cell lysate at 10 µg

Lane 4:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate at 10 µg

Lane 5:

PC-12 (Rat adrenal gland pheochromocytoma cell line) cell lysate at 10 µg

Lane 6:

NIH/3T3 (Mouse embryonic fibroblast cell line) cell lysate at 10 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 1/50000 dilution

Predicted band size: 39 kDa

Observed band size: 39 kDa

false

Exposure time: 30s