Recombinant Anti-PERP antibody [EPR7885] (ab129083)

All lanes : Anti-PERP antibody [EPR7885] (ab129083) at 1/1000 dilution

Lane 1 : Wild-type A431 cell lysate
Lane 2 : PERP knockout A431 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MOLT-4 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 21 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-PERP antibody [EPR7885] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab129083 was shown to bind specifically to PERP. A band was observed at 17 kDa in wild-type A431 cell lysates with no signal observed at this size in PERP knockout cell line ab270475 (knockout cell lysate ab270498). To generate this image, wild-type and PERP knockout A431 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes : Anti-PERP antibody [EPR7885] (ab129083) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PERP knockout HeLa cell lysate
Lane 3 : HaCaT 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: 21 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab129083 observed at 24 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab129083 Anti-PERP antibody [EPR7885] was shown to specifically react with PERP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265335 (knockout cell lysate ab258105) was used. Wild-type and PERP knockout samples were subjected to SDS-PAGE. ab129083 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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.

ab129083, at a dilution of 1/100, staining PERP in A431 cells by Immunofluorescence.

All lanes : Anti-PERP antibody [EPR7885] (ab129083) at 1/1000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Rat heart lysate
Lane 3 : A431 lysate
Lane 4 : HACAT lysate
Lane 5 : Human skin lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 21 kDa

Equilibrium disassociation constant (K_D)
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