Standard curve for TNF alpha (Analyte: ab9642); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [2C8] to TNF alpha (ab8348) at 5µg/ml and Detector Antibody Rabbit polyclonal to TNF alpha (ab9635) at 0.5µg/ml.

Chromatin was prepared from HeLa (human epithelial cell line from cervix adenocarcinoma) cells treated with and without 20 ng/ml TNF-α (ab9642) for 60 minutes according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 5 μg of ab218533 (red), and 20 µl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (SYBR green approach).

The ChIP data are consistent with the literature (PMID: 16135789).

All lanes : Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/5000 dilution

Lane 1 : WEHI-3 (Mouse leukemia lymphoblast) whole cell lysate
Lane 2 : WEHI-3 treated with 20 ng/ml TNF alpha (ab9642) for 6 h

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 80 kDa why is the actual band size different from the predicted?

All lanes : Anti-NF-kB p65 (acetyl K310) antibody [EPR21781] - ChIP Grade (ab218533) at 1/2000 dilution

Lane 1 : HEK-293 transfected with NF-kB p65 expression vector containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293 transfected with NF-kB p65 and p300 (aa1287-1663) expression vectors containing a myc-His-tag®, then treated with 20 ng/ml TNF-alpha (ab9642) for 60 minutes, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

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


Exposure time: 37 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

NF-κB p65 (acetyl K310) expression is induced by TNF-α and p300 acetyltransferases (PMID: 20160011, PMID: 12456660, PMID: 16135789).

All lanes : Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

Lane 1 : Untreated L-929 (Mouse connective tissue fibroblast cells) whole cell lysate
Lane 2 : L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (ab120382) for 8 h and then harvested.

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Observed band size: 54 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

MLKL (phospho S345) was immunoprecipitated from 1mg of L-929 (Mouse connective tissue fibroblast cells) whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic + 20 µM z-VAD compound (ab120382) for 8h using ab196436 at 1/150 dilution. Western blot was performed from the immunoprecipitate using ab196436 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h;10 µg (Input).
Lane 2: ab196436 IP in L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab196436 in L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h.

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

All lanes : Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

Lane 1 : L-929 treated with 20 ng/ml TNF alpha (ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (ab120382) for 8 h, whole cell lysate
Lane 2 : Mouse brain tissue lysate
Lane 3 : Mouse colon tissue lysate
Lane 4 : Mouse lung tissue lysate
Lane 5 : Mouse retina tissue lysate
Lane 6 : Mouse liver tissue lysate
Lane 7 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 54 kDa why is the actual band size different from the predicted?


Exposure time: 50 seconds

Blocking and diluting buffer: 5% NFDM/TBST.

MLKL pS345 is a trigger for necroptosis. It is only detectable in infection/cellular damaged (PMID:29229989) or aging tissue (PMID: 28807105) but not in normal tissues.