Anti-SIRT1 antibody [EPR18239] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

Intracellular 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

Intracellular 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

• IP

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Immunoprecipitation - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

All lanes:

Immunoprecipitation - Anti-SIRT1 antibody [EPR18239] (<a href='/en-us/products/primary-antibodies/sirt1-antibody-epr18239-ab189494'>ab189494</a>)

Predicted band size: 81 kDa

Observed band size: 110 kDa

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• WB

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Western blot - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab189494).

Lanes 1 - 6:

Western blot - Anti-SIRT1 antibody [EPR18239] (<a href='/en-us/products/primary-antibodies/sirt1-antibody-epr18239-ab189494'>ab189494</a>) at 1/1000 dilution

Lane 7:

Western blot - Anti-SIRT1 antibody [EPR18239] (<a href='/en-us/products/primary-antibodies/sirt1-antibody-epr18239-ab189494'>ab189494</a>) 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:

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 1/20000 dilution

Predicted band size: 81 kDa

Observed band size: 110 kDa,120 kDa

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• WB

Lab

Western blot - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

Western blot : Anti-SIRT1 antibody [EPR18239] (ab189494) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab189494 was shown to bind specifically to SIRT1. A band was observed at 110 kDa in wild-type A549 cell lysates with no signal observed at this size in SIRT1 knockout cell line. To generate this image, wild-type and SIRT1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-SIRT1 antibody [EPR18239] (<a href='/en-us/products/primary-antibodies/sirt1-antibody-epr18239-ab189494'>ab189494</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

SIRT1 knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293 cell lysate at 20 µg

Lane 4:

SIRT1 knockout HEK-293 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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• WB

Lab

Western blot - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

This data was developed using the same antibody clone in a different buffer formulation (ab189494).

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 CRISPR/Cas9 edited 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.

All lanes:

Western blot - Anti-SIRT1 antibody [EPR18239] (<a href='/en-us/products/primary-antibodies/sirt1-antibody-epr18239-ab189494'>ab189494</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

SIRT1 CRISPR/Cas9 edited HEK-293 cell lysate at 20 µg

Lane 3:

MDA-MB-231 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 81 kDa

Observed band size: 110 kDa

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• WB

Lab

Western blot - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (AB233398)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM /TBST Exposure time : 20,5 seconds This antibody detects strong band in testis but weak band in other tissues like colon, kidney, lymph node and liver. Please upload more lysate or use lower dilution when testing these tissues. We are unsure as to the identity of the band around 130kDa. This data was developed using the same antibody clone in a different buffer formulation (ab189494).

Lanes 1 - 6:

Western blot - Anti-SIRT1 antibody [EPR18239] - BSA and Azide free (ab233398) at 1/1000 dilution

Lanes 1 - 6:

Western blot - Anti-SIRT1 antibody [EPR18239] (<a href='/en-us/products/primary-antibodies/sirt1-antibody-epr18239-ab189494'>ab189494</a>) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Mouse colon tissue lysate at 20 µg

Lane 3:

Mouse kidney tissue lysate at 20 µg

Lane 4:

Mouse lymph node tissue lysate at 20 µg

Lane 5:

Mouse liver tissue lysate at 20 µg

Lane 6:

Mouse liver tissue lysate

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 1/20000 dilution

Predicted band size: 81 kDa

Observed band size: 120 kDa,110 kDa,75 kDa

false

Exposure time: 20.5s