Anti-FADS2 antibody

• IP

Unknown

Immunoprecipitation - Anti-FADS2 antibody (AB72189)

FADS2 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Rabbit polyclonal to FADS2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab72189.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 45kDa : FADS2.

All lanes:

Immunoprecipitation - Anti-FADS2 antibody (ab72189)

Predicted band size: 52 kDa

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• WB

Lab

Western blot - Anti-FADS2 antibody (AB72189)

Western blot : Anti-FADS2 antibody (ab72189) staining at 1 ug/ml, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab72189 was shown to bind specifically to FADS2. A band was observed at 40-45 kDa in wild-type A549 cell lysates with no signal observed at this size in FADS2 knockout cell line. To generate this image, wild-type and FADS2 knockout A549 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-FADS2 antibody (ab72189) at 1 µg/mL

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human FADS2 Knockout A549 cell line (<a href='/ko/products/cell-lines/human-fads2-knockout-a549-cell-line-ab324211'>ab324211</a>) 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

Predicted band size: 52 kDa

Observed band size: 40 kDa,45 kDa

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• WB

Unknown

Western blot - Anti-FADS2 antibody (AB72189)

ab72189 detects a strong band at 45 kDa in human liver and brain tissue lysates, aswell as hela and hepG2 whole cell lysates. This antibody was raised against an immunogen that is predicted to cross react with all known isoforms of Fatty acid desaturase 2 (FADS2). FADS2 has three isoforms, isoform one has a molecular weight of 52 kDa with isoforms two and three a molecular weight of 49 and 45 respectively(SwissProt data). The band we have observed is thought to represent isoform 3 of FADS2 (45 kDa).

All lanes:

Western blot - Anti-FADS2 antibody (ab72189) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Human liver tissue lysate - total protein (<a href='/ko/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

Human brain tissue lysate - total protein (<a href='/ko/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 52 kDa

Observed band size: 45 kDa,62.5 kDa

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Exposure time: 2min