All lanes : Anti-TEF1/TEAD-1 antibody [EPR3967(2)] (ab133533) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : TEAD1 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 52 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab133533 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab133533 was shown to react with TEF1/TEAD-1 in wild-type A549 cells in western blot with loss of signal observed in TEAD1 knockout sample. Wild-type and TEAD1 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab133533 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue sections labeling TEF1/TEAD-1 with Purified ab133533 at 1:1000 dilution (1.63 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Anti-TEF1/TEAD-1 antibody [EPR3967(2)] (ab133533) at 1/2000 dilution (purified) + Human fetal muscle lysates at 15 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 52 kDa
Observed band size: 52 kDa

Blocking and diluting buffer: 5% NFDM/TBST

ab133533 (purified) at 1:80 dilution (2ug) immunoprecipitating TEF1/TEAD-1 in 293 (Human embryonic kidney epithelial cell) whole cell lysate.
Lane 1 (input): 293 (Human embryonic kidney epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab133533 & 293 (Human embryonic kidney epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab133533 in 293 (Human embryonic kidney epithelial cell) whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue sections labeling TEF1/TEAD-1 with Purified  ab133533 at 1:1000 dilution (1.63 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue sections labeling TEF1/TEAD-1 with Purified  ab133533 at 1:1000 dilution (1.63 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry analysis of Paraffin Embedded Human placenta tissue labelling TEF1/TEAD-1 with unpurified ab133533 at 1/100. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-TEF1/TEAD-1 antibody [EPR3967(2)] (ab133533) at 1/10000 dilution (purified)

Lane 1 : L6 ( Rat skeletal muscle myoblast) whole cell lysates
Lane 2 : C2C12 ( Mouse myoblasts myoblast) whole cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 52 kDa
Observed band size: 52 kDa

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-TEF1/TEAD-1 antibody [EPR3967(2)] (ab133533) at 1/1000 dilution (unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : 293T cell lysate
Lane 3 : Fetal muscle lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 52 kDa

Immunohistochemistry analysis of Paraffin Embedded Human skeletal muscle tissue labelling TEF1/TEAD-1 with unpurified ab133533 at 1/100. Heat mediated antigen retrieval was performed using citrate buffer pH 6 before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
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