All lanes : Anti-NDUFS3 antibody [EPR12781] (ab183733) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : NDUFS3 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 30 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab183733 observed at 27 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab183733 was shown to react with NDUFS3 in HEK-293T wild-type cells in western blot with loss of signal observed in NDUFS3 knockout cell line ab266419 (NDUFS3 knockout cell lysate ab257556). Wild-type and NDUFS3 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab183733 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 10000 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 hepatocellular carcinoma tissue labeling NDUFS3 with ab183733 at 1/50 dilution followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

All lanes : Anti-NDUFS3 antibody [EPR12781] (ab183733) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : NDUFS3 knockout HEK-293T cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HL60 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 30 kDa
Observed band size: 30 kDa

Lanes 1- 4: Merged signal (red and green). Green - ab183733 observed at 30 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab183733 was shown to react with NDUFS3 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266419 (knockout cell lysate ab257556) was used. Wild-type HEK-293T and NDUFS3 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab183733 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-NDUFS3 antibody [EPR12781] (ab183733) at 1/100000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal brain lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 30 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

All lanes : Anti-NDUFS3 antibody [EPR12781] (ab183733) at 1/20000 dilution

Lane 1 : C6 cell lysate
Lane 2 : Raw264.7 cell lysate
Lane 3 : PC-12 cell lysate
Lane 4 : NIH/3T3 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 30 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?