Human LOXL2 knockout HeLa cell lysate

• WB

Lab

Western blot - Human LOXL2 knockout HeLa cell lysate (AB257168)

Lane 1 : Wild-type HeLa cell lysate (20 μg)

Lane 2 : LOXL2 knockout HeLa cell lysate (20 μg)

Lane 3 : HeLa treated with 0.5nM CoCl2 for 6 hours whole cell lysate (20 μg)

Lane 4 : Untreated HeLa cell lysate (20 μg)

Lanes 1-4 : Merged signal (red and green). Green - ab179810 observed at 105 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab179810 Anti-LOXL2 antibody [EPR12733] - C-terminal was shown to specifically react with LOXL2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261804 (knockout cell lysate ab257168) was used. Wild-type and LOXL2 knockout samples were subjected to SDS-PAGE. ab179810 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-LOXL2 antibody [EPR12733] - C-terminal (<a href='/en-us/products/primary-antibodies/loxl2-antibody-epr12733-c-terminal-ab179810'>ab179810</a>) at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

LOXL2 knockout HeLa cell lysate at 20 µg

Lane 4:

Untreated HeLa cell lysate at 20 µg

Predicted band size: 87 kDa

Observed band size: 105 kDa

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• Sanger seq

Unknown

Sanger Sequencing - Human LOXL2 knockout HeLa cell lysate (AB257168)

Allele-1 : 1 bp insertion in exon 4

• Sanger seq

Unknown

Sanger Sequencing - Human LOXL2 knockout HeLa cell lysate (AB257168)

Allele-4 :

• Sanger seq

Unknown

Sanger Sequencing - Human LOXL2 knockout HeLa cell lysate (AB257168)

Allele-3 :

• Sanger seq

Unknown

Sanger Sequencing - Human LOXL2 knockout HeLa cell lysate (AB257168)

Allele-2 :

• WB

Supplier Data

Western blot - Human LOXL2 knockout HeLa cell lysate (AB257168)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS Negative control : MCF7 (PMID : 19330836). Lysates at 20 µg per lane. The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-LOXL2 antibody (ab314140) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab314140 was shown to bind specifically to LOXL2. Target of interest was observed at 87 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in LOXL2 knockout cell line (lane 2, knockout cell line ab261804 / knockout cell lysate ab257168). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane.  Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C.  Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-LOXL2 antibody [EPR28298-7] (<a href='/en-us/products/primary-antibodies/loxl2-antibody-epr28298-7-ab314140'>ab314140</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa whole cell lysate at 20 µg

Lane 2:

Western blot - Human LOXL2 knockout HeLa cell lysate (ab257168) at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 87 kDa

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