重组人TNF alpha蛋白
参阅全部 TNF alpha 蛋白酶
The ED50, as determined by the cytolysis of murine L929 cells in the presence of Actinomyocin D, is ≤ 0.05 ng/mL, corresponding to a specific activity of ≥ 2 x 107 units/mg.
> 98 % SDS-PAGE.
>98% by HPLC analyses. Sterile filtered.
< 1.000 Eu/µg
Escherichia coli
Full length protein
No
Recombinant
Human
VRSSSRTPSD KPVAHVVANP QAEGQLQWLN RRANALLANG VELRDNQLVV PSEGLYLIYS QVLFKGQGCP STHVLLTHTI SRIAVSYQTK VNLLSAIKSP CQRETPEGAE AKPWYEPIYL GGVFQLEKGD RLSAEINRPD YLDFAESGQV YFGIIAL
17 kDa
77 to 233
aa 77 to 233 refers to the full length mature form (soluble).
Our Abpromise guarantee covers the use of ab9642 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
应用
Functional Studies
Sandwich ELISA
HPLC
SDS-PAGE
Lyophilized
Lots prior to June 2015 contain 0.036% Tris (including lots GR157466-11, -12 and -13).
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
Constituents: 0.049% Sodium phosphate, 0.12% Sodium chloride
This product is an active protein and may elicit a biological response in vivo, handle with caution.
复溶
Reconstitute with dH2O to make a final concentration between 0.1 to 1.0 mg/ml.
APC1
APC1 protein
Cachectin
DIF
Differentiation inducing factor
Macrophage cytotoxic factor
Tnf
TNF superfamily member 2
TNF superfamily, member 2
TNF, macrophage derived
TNF, monocyte derived
TNF-a
TNF-alpha
TNFA
TNFA_HUMAN
TNFSF2
Tumor necrosis factor
Tumor necrosis factor (TNF superfamily member 2)
Tumor necrosis factor alpha
Tumor necrosis factor ligand superfamily member 2
Tumor Necrosis Factor, Membrane Form
Tumor necrosis factor, soluble form
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 - 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).
Western blot - Recombinant human TNF alpha protein (ab9642)
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 kDawhy is the actual band size different from the predicted?
Western blot - Recombinant human TNF alpha protein (ab9642)
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 kDawhy 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).
Western blot - Recombinant human TNF alpha protein (ab9642)
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 kDawhy is the actual band size different from the predicted?
Exposure time: 15 seconds
Blocking and dilution buffer: 5% NFDM/TBST.
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.
Western blot - Recombinant human TNF alpha protein (ab9642)
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 kDawhy 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.
