All lanes : Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SIRT6 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 39 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?

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.

 

 

Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/10000 dilution + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 39 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

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.

Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/10000 dilution + Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 39 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

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.

Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/10000 dilution + NIH/3T3 (Mouse embryonic fibroblast cell line) cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 39 kDa
Observed band size: 39 kDa


Exposure time: 30 seconds

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

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cytoplasmic lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) nuclear lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 39 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

SIRT6 is detected in nuclear fractions.

All lanes : Anti-SIRT6 antibody [EPR18463] (ab191385) at 1/2000 dilution

Lane 1 : Rat brain lysate
Lane 2 : Rat spleen lysate
Lane 3 : C6 (Rat glial tumor cell line) cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate
Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma cell line) cell lysate
Lane 6 : NIH/3T3 (Mouse embryonic fibroblast cell line) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 39 kDa
Observed band size: 39 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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.

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.

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.