All lanes : Anti-SIRT1 antibody [19A7AB4] (ab110304) at 1 µg/ml

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: 81 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 - ab110304 observed at 110 kDa. Red - loading control, ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab110304 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 ab110304 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) 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 testis* 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 ab110304, 1ug/ml, 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

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 ab110304, 5ug/ml, 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

Immunocytochemistry/Immunofluorescence analysis using ab110304 at 0.5µg/ml staining SIRT1 in HDFn cells (paraformaldehyde fixed and Triton X-100 permeabilized). The secondary antibody was (Red) Alexa Fluor® 594 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. 10% Goat serum was used as the blocking agent for all blocking steps.

HL-60 cells were stained with 1 µg/mL SIRT1 antibody ab110304(blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.

All lanes : Anti-SIRT1 antibody [19A7AB4] (ab110304) at 1 µg/ml

Lane 1 : HeLa whole cell lysate
Lane 2 : HepG2 whole cell lysate
Lane 3 : F9 whole cell lysate
Lane 4 : MEF1 whole cell lysate
Lane 5 : PC-12 whole cell lysate
Lane 6 : RBL-1 whole cell lysate
Lane 7 : Human Testis tissue lysate
Lane 8 : Human Colon tissue lysate
Lane 9 : Mouse Testis tissue lysate
Lane 10 : Mouse Colon tissue lysate
Lane 11 : Rat Testis tissue lysate
Lane 12 : Rat Colon tissue lysate

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 81 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 4 minutes

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 ab110304 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.

All lanes : Anti-SIRT1 antibody [19A7AB4] (ab110304) at 1/1000 dilution

All lanes : rat Skeletal muscle

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Mouse at 1/6000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 81 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?


Exposure time: 13 minutes

Blocked with 3% milk (TBS-tween) at 4C for 16 hours

See Abreview

All lanes : Anti-SIRT1 antibody [19A7AB4] (ab110304) at 0.125 µg/ml

Lane 1 : HepG2 cell lysate(Human)
Lane 2 : H9C2 cell lysate (Rat)
Lane 3 : MEF cell lysate (Mouse)

Predicted band size: 81 kDa

WB Conditions
Primary Antibody: 0.25 µg/mL in 10X Blocking Buffer (ab126587). 3hrs at room temperature.

Secondary Antibody: 1:5,000  in 10X Blocking Buffer (ab126587). 3hrs at room temperature.

ab110304 detects a band of approximately 110 kDa (110-121 kDa) which is likely to be due to post translational glycosylation. SIRT1 is known to bind to several other proteins, and the 121kDa band could also be due to the presence of one of these complexes (ensure samples are adequately reduced and denatured).