Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26]

• IP

Supplier Data

Immunoprecipitation - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (AB307889)

Cytosolic Phospholipase A2 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug with ab307889 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307889 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HeLa whole cell lysate 10 ug Lane 2 : ab307889 IP in HeLa whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307889 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 50 seconds.

All lanes:

Immunoprecipitation - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (ab307889) at 1/30 dilution

All lanes:

ab307889 IP in HeLa whole cell lysate at 10 µg

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Exposure time: 50s

• WB

Supplier Data

Western blot - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (AB307889)

In lane 1, the lysate was freshly made and used for Western Blotting immediately to minimize protein degradation. In lanes 2-3, the lysates were stored at -80℃ prior to Western Blotting. The bands beneath the target band (75 kDa) are likely to be degradation products. The secondary antibody dilution for Lane1 was 1 : 100000, the secondary antibody dilution for Lane2 and 3 was 1 : 20000. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (ab307889) at 1/1000 dilution

Lane 1:

HeLa whole cell lysate at 20 µg

Lane 2:

HeLa transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 3:

HeLa transfected with siRNA specifically targeting Cytosolic Phospholipase A2 whole cell lysate at 20 µg

Secondary

Lane 1:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lanes 2 - 3:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 100 kDa

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• WB

Supplier Data

Western blot - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (AB307889)

Lysates at 20 µg per lane. The samples were run on a Bis-Tris gel. Performed under reducing conditions. False colour image of Western blot : Anti-Cytosolic Phospholipase A2 antibody (ab307889) 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, ab307889 was shown to bind specifically to Cytosolic Phospholipase A2. A band was observed at 100 kDa in wild-type HAP1 cell lysates with no signal observed at this size in Cytosolic Phospholipase A2 knockout cell line. To generate this image, wild-type and Cytosolic Phospholipase A2 knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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. The identity of the lower MW band at approximately 75 kDa is unknown.

All lanes:

Western blot - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (ab307889) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Cytosolic Phospholipase A2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

A549 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 85 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (AB307889)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602).

In Western blot, ab307889 was shown to bind specifically to Cytosolic Phospholipase A2. A band was observed at 100 kDa in wild-type HeLa cell lysates with no signal observed at this size in Cytosolic Phospholipase A2 knockout cell line(lane 2) (lane 2, knockout cell line ab265490 / knockout cell lysate ab257133).

All lanes:

Western blot - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (ab307889) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Cytosolic Phospholipase A2 knockout HeLa whole cell lysate at 20 µg

Lane 2:

Western blot - Human PLA2G4A (Cytosolic Phospholipase A2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-pla2g4a-cytosolic-phospholipase-a2-knockout-hela-cell-line-ab265490'>ab265490</a>)

Lane 2:

Western blot - Human PLA2G4A (Cytosolic Phospholipase A2) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-pla2g4a-cytosolic-phospholipase-a2-knockout-hela-cell-lysate-ab257133'>ab257133</a>) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 100 kDa

false

Exposure time: 38s

• WB

Supplier Data

Western blot - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (AB307889)

Lane 3 was developed using a high sensitivity ECL substrate. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cytosolic Phospholipase A2 antibody [EPR26068-26] (ab307889) at 1/1000 dilution

Lane 1:

NIH/3T3 whole cell lysate at 20 µg

Lane 2:

RAW264.7 whole cell lysate at 20 µg

Lane 3:

C6 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 85 kDa

Observed band size: 100 kDa

true