All lanes : Anti-SIRT1 antibody [EPR18239] (ab189494) at 1/1000 dilution

Lane 1 : Wild-type HEK-293 cell lysate
Lane 2 : SIRT1 knockout HEK-293 cell lysate
Lane 3 : MDA-MB-231 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 80 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab189494 observed at 110 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab189494 was shown to react with SIRT1 in western blot. The band observed in the knockout lysate lane below 110kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab189494 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling SIRT1 with ab189494 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining in human skeletal muscle (PMID: 23332867) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat-mediated antigen retrieval using Citrate, pH 6.0.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling SIRT1 with ab189494 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 nuclear and weakly cytoplasmic staining in HeLa cell line.

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

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% methanol-permeabilized F9 (mouse embryonic testicular cancer cell line) cells labeling SIRT1 with ab189494 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 nuclear and weakly cytoplasmic staining in F9 cell line.

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

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.

 

Lanes 1-6 : Anti-SIRT1 antibody [EPR18239] (ab189494) at 1/1000 dilution
Lane 7 : Anti-SIRT1 antibody [EPR18239] (ab189494) at 1/5000 dilution

Lane 1 : Mouse testis tissue lysate at 20 µg
Lane 2 : F9 (mouse embryonic testicular cancer cell line) whole cell lysate at 20 µg
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 4 : Rat E18 brain tissue lysate at 20 µg
Lane 5 : A549 (human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 6 : Human testis tissue lysate at 10 µg
Lane 7 : Rat testis tissue lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Developed using the ECL technique.

Predicted band size: 80 kDa
Observed band size: 110,120 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times:

Lanes 1 & 2: 8 seconds

Lane 3: 32 seconds

Lanes 4 & 5: 67 seconds

Lane 6: 59 seconds

Lane 7: 10 seconds

The expression profile is consistent with what has been described in the literature (PMID: 21474819).

The 75 kDa band is a cleaved fragment of SIRT1 (PMID: 25770475, PMID: 21305533), while the approximately 85 kDa band likely represents a splice variant (PMID: 20975832).

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling SIRT1 with ab189494 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Mainly nuclear staining in human testis (PMID: 17197703) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat-mediated antigen retrieval using Citrate, pH 6.0.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling SIRT1 with ab189494 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Mainly nuclear staining in mouse testis (PMID: 17197703) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat-mediated antigen retrieval using Citrate, pH 6.0.

Immunohistochemical analysis of paraffin-embedded rat skeletal muscle tissue labeling SIRT1 with ab189494 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining in rat skeletal muscle (PMID: 23332867) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat-mediated antigen retrieval using Citrate, pH 6.0.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling SIRT1 with ab189494 at 1/60 dilution (red) compared with a rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled 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.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized F9 (mouse embryonic testicular cancer cell line) cell line labeling SIRT1 with ab189494 at 1/60 dilution (red) compared with a rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled 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.

SIRT1 was immunoprecipitated from 0.35 mg of F9 (mouse embryonic testicular cancer cell line) whole cell lysate with ab189494 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab189494 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: F9 whole lysate 10 μg (Input).
Lane 2: ab189494 IP in F9 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab189494 in F9 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 50 seconds.