All lanes : Anti-SIRT1 antibody (ab7343) 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: 81.3 kDa
Observed band size: 105 kDa why is the actual band size different from the predicted?

This product was validated with an edited cell line that does not express the epitope recognised by this antibody. Testing with other antibodies has shown that the edited cell line produces a truncated protein.

Lanes 1 - 4: Merged signal (red and green). Green - ab7343 observed at 105 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab7343 was shown to react with SIRT1 in wild-type HEK-293 cells in Western blot with loss of signal observed in SIRT1 knockout sample. Wild-type HEK-293 and SIRT1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab7343 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml 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 h at room temperature before imaging.

All lanes : Anti-SIRT1 antibody (ab7343) at 1 µg/ml

Lane 1 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate - Vector Transfected Control
Lane 2 : HEK293 Whole Cell Lysate Transiently Overexpressing SIRT1

Lysates/proteins at 5 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 81.3 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

SIRT1 was immunoprecipitated from HeLa nuclear extract. The  IP'd fraction was incubated with a histone H4 peptide acetylated with tritiated acetyl-coA, in the presence or absence of NAD+ (the cofactor for SIRT's). After incubation the free label (released acetyl groups) was separated from the peptide.

40ug of whole cell extract (over-expressing the SIRT1 protein) is probed with anti-SIRT1 antibody (ab7343).

The protein has not been detected in Western blot in HeLa lysates.

40ug of whole cell extract (over-expressing the SIRT1 protein) is probed with anti-SIRT1 antibody (ab7343). The protein has not been detected in Western blot in HeLa lysates.