Recombinant human TNF alpha protein

• IP

Lab

Immunoprecipitation - Recombinant human TNF alpha protein (AB9642)

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:

Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (<a href='/products/primary-antibodies/mlkl-phospho-s345-antibody-epr95152-ab196436'>ab196436</a>)

All lanes:

Immunoprecipitation - Recombinant human TNF alpha protein (ab9642)

Predicted band size: 54 kDa

Observed band size: 54 kDa

false

• sELISA

Supplier Data

Sandwich ELISA - Recombinant human TNF alpha protein (AB9642)

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.

• ChIP

Supplier Data

ChIP - Recombinant human TNF alpha protein (AB9642)

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).