All lanes : Anti-SIRT1 antibody [E104] (ab32441) at 1/20000 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: 82 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 - ab32441 observed at 110 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab32441 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 ab32441 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 20000 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.

IHC image of SIRT1 staining in a section of formalin-fixed paraffin-embedded normal human colon* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab32441, 1/250 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Immunofluorescence staining of SH-SY5Y cells with purified ab32441 at a working dilution of 1 in 150, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti rabbit (ab150077), used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab32441 was used at a dilution of 1/200 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500.

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling SIRT1 (red) with ab32441 at a 1/200 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

All lanes : Anti-SIRT1 antibody [E104] (ab32441) at 1/20000 dilution (purified)

Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : HEK293 cell lysate
Lane 4 : A549 cell lysate
Lane 5 : SW480 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 82 kDa
Additional bands at: 110 kDa (possible glycosylated form)

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

ab32441 (purified) at 1/30 immunoprecipitating SIRT1 in Jurkat cells (Lane 1). For western blotting, a HRP-conjugated anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-SIRT1 antibody [E104] (ab32441) at 1/20000 dilution

Lane 1 : HeLa whole cell lysate
Lane 2 : HepG2 whole cell lysate
Lane 3 : Human Testis tissue lysate
Lane 4 : Human Colon tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 82 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 150 seconds

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab32441 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

IHC image of SIRT1 staining in a section of formalin-fixed paraffin-embedded normal human colon* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab32441, 1/250 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab32441 at a working dilution of 1 in 150. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using unpurified ab32441 at 1/100 dilution.

Immunohistochemical analysis of paraffin-embedded human lung squamous carcinoma using unpurified ab32441 at 1/100 dilution.