Human TNF-alpha Recombinant (E.Coli)

Description

Methods (16)


accession	P01375

Source	Optimized DNA sequence encoding Human Tumor Necrosis Factor-alpha mature chain was expressed in Escherichia Coli.
Molecular weight	Native human Tumor Necrosis Factor-alpha is generated by the proteolytic removal of the signal peptide and propeptide, this molecule has a calculated mass of approximately 17kDa. Recombinant TNF-alpha is a monomer protein,consisting of 158 amino acids and migrates as an approximately 17 kDa protein under reducing conditions.
Purity	>95%, as determined by SDS-PAGE and HPLC
Biological Activity	The ED(50) was determined cytolysis of murine L929 cells in the presence of Actinomycin D is ≤.0.1 ng/ml, corresponding to a specific activity of >1x10⁷ units/mg.
Protein Sequence	MSTESMIRDV ELAEEALPKK TGGPQGSRRC LFLSLFSFLI VAGATTLFCL LHFGVIGPQR EEFPRDLSLI SPLAQAVRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELR DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL
Endotoxin	Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation	TNF-a was lyophilized from a.2μm filtered concentrated (1mg/ml) solution in PBS, pH.2.
Reconstitution	A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers.
Storage	The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage	This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.

Interactor	O88351
Interactor	O88522
Interactor	O89110
Interactor	P01374 TNFB_HUMAN
Interactor	P01730 CD4_HUMAN
Interactor	P25118 TNR1A_MOUSE
Interactor	P39429
Interactor	P62991
Interactor	Q12933
Interactor	Q15628
Interactor	Q60680
Interactor	Q60769
Interactor	Q60855
Interactor	Q62073
Interactor	Q924T7
Interactor	Q9QZL0
Interactor	P19438 TNR1A_HUMAN
Interactor	P20333 TNR1B_HUMAN
Interactor	P25118 TNR1A_MOUSE
Interactor	P36941 TNR3_HUMAN
Interactor	P70191
Interactor	Q13546
Interactor	Q3U0V2
Interactor	Q80TQ2
Interactor	Q9DHW0
Interactor	Q9QZL0
Molecular function	Cytokine

Methods


Urokinase-Type Plasminogen Activator (uPA)-Plasmingen Assay MDCK (1.5 × 10³ cells per well) and SMMC-7721 (6 × 10³ cells per well) cells stably overexpressing an empty vector or pIgR cDNA were seeded in 96-well plates and grown overnight at 37°C. Then cells were cultured in fresh medium for 24 hours. Alternatively, HT29 cells were seeded at 6000 cells per well in 96-well plates and grown overnight at 37°C. Fresh medium containing tumor necrosis factor α (TNF-α) (5, 10, 20 ng/mL) was added to the appropriate wells and incubated for 24 hours at 37°C. The plates were processed for determination of plasminogen activity by first rinsing twice with phenol red–free DMEM and then adding 200 μL of reaction buffer containing 50% (vol/vol) 0.05 units/mL plasminogen in phenol red–free DMEM, 40% (vol/vol) 50 mM Tris–HCl pH 8.2, 10% (vol/vol) 3 mM Chromozyme PL , and 0.2% tween 20… MDCK (1.5 × 10³ cells per well) and SMMC-7721 (6 × 10³ cells per well) cells stably overexpressing an empty vector or pIgR cDNA were seeded in 96-well plates and grown overnight at 37°C. Then cells were cultured in fresh medium for 24 hours. Alternatively, HT29 cells were seeded at 6000 cells per well in 96-well plates and grown overnight at 37°C. Fresh medium containing tumor necrosis factor α (TNF-α) (5, 10, 20 ng/mL) was added to the appropriate wells and incubated for 24 hours at 37°C. The plates were processed for determination of plasminogen activity by first rinsing twice with phenol red–free DMEM and then adding 200 μL of reaction buffer containing 50% (vol/vol) 0.05 units/mL plasminogen in phenol red–free DMEM, 40% (vol/vol) 50 mM Tris–HCl pH 8.2, 10% (vol/vol) 3 mM Chromozyme PL , and 0.2% tween 20 in 100 mM glycine solution to each well. The plates were incubated for 6 hours at 37°C, 5% CO₂, and the absorbance of each well was read using an automated spectrophotometric plate reader at 405 nm. The results were repeated in three independent experiments. Read more
Effects of VEGF and TNFá on Tie1 and Tie2 Cleavage. HUVEC were incubated with VEGF A or TNFα B for times up to 24 h as indicated before cell lysis and detection of cellular full-length Tie1 by immunoblotting.
Annexin V staining Non-silencing and knock-down cells were plated at equal density and treated with TNFα (1000ng/ml) (300-01A) where indicated. After 8h of TNFα treatment, the Annexin V staining assay was performed as described in the user manual (TACS Annexin V 4830-01-K R& ) then samples were subjected to flow cytometry analysis .
2.5. DC Maturation and Rapamycin Treatment Two days after monocytes were plated, monocyte-derived and hESC-derived immature DC were treated with 10 ng/mL and 5–7 ng/mL of rapamycin , respectively. On day 5, Cs were matured for 48 hr using a maturation cocktail consisting of 50 ng/mL of GM-CSF , 100 ng/mL IL-4 , 20 ng/mL IFNγ , 50 ng/mL TNFα , 10 ng/mL of IL-1β , and 1 μg/mL PGE₂. On day 6-7, DCs were harvested by gentle pipetting, passed through a 70 μm cell strainer, centrifuged, and resuspended prior to their use in experiments.
Generation of DC Blood samples from healthy donors are collected after informed consent, in accordance with the Declaration of Helsinki and approval of the of the University Hospital of the Ludwig-Maximilians-University, Munich, Germany. Peripheral blood mononuclear cells (PBMC) are isolated by Ficoll density gradient centrifugation. PBMC are resuspended in 15 ml VLE (very low endotoxin) medium'>RPMI 1640 medium supplemented with 1.5% human serum medium'>(DC medium) at 7.5′10⁷ cells per 75 cm² culture flask (NUNC, 178905) and incubated at 37°C and 5% CO₂ for 1 h. Non-adherent cells are carefully removed by washing. Adherent monocytes are cultured in medium containing 100 ng/ml GM-CSF (Leukine^® by Berlex, NC50419-050-30) and 20 ng/ml interleukin-4 (& 104-IL-050-CF) and fed with the same medium on days 3 and 6. On day 6 of culture, the immature C are differentiated into mC by addition of medium containing 10 ng/ml IL-1β… Blood samples from healthy donors are collected after informed consent, in accordance with the Declaration of Helsinki and approval of the of the University Hospital of the Ludwig-Maximilians-University, Munich, Germany. Peripheral blood mononuclear cells (PBMC) are isolated by Ficoll density gradient centrifugation. PBMC are resuspended in 15 ml VLE (very low endotoxin) medium'>RPMI 1640 medium supplemented with 1.5% human serum medium'>(DC medium) at 7.5′10⁷ cells per 75 cm² culture flask (NUNC, 178905) and incubated at 37°C and 5% CO₂ for 1 h. Non-adherent cells are carefully removed by washing. Adherent monocytes are cultured in medium containing 100 ng/ml GM-CSF (Leukine^® by Berlex, NC50419-050-30) and 20 ng/ml interleukin-4 (& 104-IL-050-CF) and fed with the same medium on days 3 and 6. On day 6 of culture, the immature C are differentiated into mC by addition of medium containing 10 ng/ml IL-1β (& 201-LB-025-CF), 15 ng/ml IL-6 (& 206-IL-050-CF), 10 ng/ml TNFα (& 210-TA-050-CF) and 1 µg/ml PGE₂ for 2 d. Read more
Hyperthermia increases viral reactivation in J-Lat 10.6 cells. Stimulation of J-Lat 10.6 cells with TNFα triggers HIV-1 reactivation and GFP expression.
Treatment of Cell Cultures Human recombinant (r)IL-1ß (, , 10 ng/ml) was applied and maintained for different time periods (from 10 min to 48 h) before harvesting the cells for RNA isolation or western blot analysis. Medium was collected to perform enzyme-linked immunosorbent assay (ELISA). In some experiments lipo-polysaccharide (LPS; 100 ng/ml, , ), rIL-6 (10 ng/ml; athmann , , ), tumor necrosis factor α (TNFα; 1 ng/ml, , ) and High mobility group box 1 (HMGB1; 40 nM; HMGh , , ) alone or together with IL-1 ß were applied and maintained in the medium for 24 before harvesting the cells for RNA isolation. In some experiments cells stimulated with LPS were treated with LPS-RS (a TLR4 antagonist from the photosynthetic bacterium Rhodobacter sphaeroides, , 10 µg/ml), applied 1 h before LPS. Human IL-1 receptor antagonist (IL-1Ra; 1 µg/ml, , ) was used to neutralize IL-1β activity (applied 1 h before IL-1β). As previously…
Macrophage-secreted TNFα enhances breast fibroblast proliferation. Primary human breast fibroblasts were placed in 2% charcoal-stripped serum-containing media 24 hr prior to treatment with 2% charcoal-stripped serum-containing media + estrogen (10-10 M), media + 10 ng/ml TNFα , + TNFα inhibitor , the combination TNFα + inhibitor , macrophage-conditioned media or CM + TNFα inhibitor .
Treatment of cell cultures Human recombinant (r)IL-1β (, , 10 ng/mL) was applied and maintained for 24 h before harvesting the cells for RNA isolation, western blot analysis or for immunocytochemistry. In some experiments different time periods of IL-1β exposure (ranging from 10 min to 48 h) were used and rIL-6 (10 ng/mL , , ), tumor necrosis factor α (TNFα; 1 ng/mL, , ) and high mobility group box 1 (HMGB1; 40nM; HMGh , , ) alone or together with IL-1β were applied and maintained in the medium for 24 h before harvesting the cells for RNA isolation. Human IL-1receptor antagonist (IL-1Ra; 1 μg/mL, , ) was used to neutralize IL-1β activity (applied 1 h before IL-1β). As previously shown [
Preparation of DCs and CD8+ T cells Cs were generated as described with some modifications (⁺ T cells and the adherent cells were cultured for 7 days in medium'>PMI-1640 medium containing 10% FCS, 800 U/ml recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and 500 U/ml recombinant human interleukin-4 (IL-4) (both from& , , ) for generating Cs. The media along with the necessary cytokines were half-refreshed every other day and 50 U/ml tumor necrosis factor-α (TNF-α) was added to the culture medium on the sixth day.

Human TNF-alpha Recombinant (E.Coli)

Human TNF-alpha Recombinant (E.Coli)

Description

Methods (16)

Methods

Urokinase-Type Plasminogen Activator (uPA)-Plasmingen Assay

Effects of VEGF and TNFá on Tie1 and Tie2 Cleavage.

Annexin V staining

2.5. DC Maturation and Rapamycin Treatment

Generation of DC

Hyperthermia increases viral reactivation in J-Lat 10.6 cells.

Treatment of Cell Cultures

Macrophage-secreted TNFα enhances breast fibroblast proliferation.

Treatment of cell cultures

Preparation of DCs and CD8+ T cells

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