All lanes : Anti-SNAP29 antibody [EPR9199] (ab138500) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SNAP29 knockout HeLa cell lysate
Lane 3 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 27 kDa
Observed band size: 29 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab138500 observed at 29 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab138500 Anti-SNAP29 antibody [EPR9199]  was shown to specifically react with SNAP29 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265289 (knockout cell lysate ab257693) was used.  Wild-type and SNAP29 knockout samples were subjected to SDS-PAGE.  ab138500 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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-SNAP29 antibody [EPR9199] (ab138500) at 1/2000 dilution (purified)

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 27 kDa
Observed band size: 29 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

All lanes : Anti-SNAP29 antibody [EPR9199] (ab138500) at 1/2000 dilution (purified)

Lane 1 : HeLa cell lysate
Lane 2 : HEK293 cell lysate
Lane 3 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 27 kDa
Observed band size: 29 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

ab138500 (purified) at 1/20 immunoprecipitating SNAP29 in 10 μg HeLa cell lysate (Lanes 1 and 2, observed at 29 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730). For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution. Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

All lanes : Anti-SNAP29 antibody [EPR9199] (ab138500) at 1/1000 dilution (unpurified)

Lane 1 : 293T cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 27 kDa
Observed band size: 29 kDa why is the actual band size different from the predicted?

Equilibrium disassociation constant (K_D)
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