Recombinant Anti-Leukotriene A4 hydrolase/LTA4H antibody [EPR5713] (ab133512)

All lanes : Anti-Leukotriene A4 hydrolase/LTA4H antibody [EPR5713] (ab133512) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : LTA4H knockout HEK293T cell lysate
Lane 3 : T-47D cell lysate
Lane 4 : A549 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: 69 kDa
Observed band size: 69 kDa

Lanes 1-4: Merged signal (red and green). Green - ab133512 observed at 69 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab133512 Anti-Leukotriene A4 hydrolase/LTA4H antibody [EPR5713] was shown to specifically react with Leukotriene in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266467 (knockout cell lysate ab258034) was used. Wild-type and Leukotriene knockout samples were subjected to SDS-PAGE. ab133512 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.

Overlay histogram showing Jurkat cells stained with ab133512 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab133512, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

 

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling Leukotriene A4 hydrolase/LTA4H with ab133512 at 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-Leukotriene A4 hydrolase/LTA4H antibody [EPR5713] (ab133512) at 1/10000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : 293T cell lysate
Lane 3 : T47D cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 69 kDa

All lanes : Anti-Leukotriene A4 hydrolase/LTA4H antibody [EPR5713] (ab133512) at 1/5000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart tissue lysate
Lane 3 : Mouse kidney tissue lysate
Lane 4 : Mouse spleen tissue lysate
Lane 5 : Rat brain tissue lysate
Lane 6 : Rat heart tissue lysate
Lane 7 : Rat kidney tissue lysate
Lane 8 : Rat spleen tissue lysate

Predicted band size: 69 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Equilibrium disassociation constant (K_D)
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