Rat Tumor Necrosis Factor-alpha Recombinant

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Rat Tumor Necrosis Factor-alpha Recombinant

$70.00$2,700.00


accession P16599


Source Optimized DNA sequence encoding Rat Tumor Necrosis Factor-alpha mature chain was expressed in Escherichia Coli.
Molecular weight Native Rat TNF-alpha, generated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calculated molecular mass of approximately 17kDa. Recombinant Rat Tumor Necrosis Factor-alpha is a monomeric protein consisting of 157 amino acid residue subunits, and migrates as an approximately 17 kDa protein under non-reducing conditions and reducing conditions in SDS-PAGE.
Purity >98%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) was determined by the dose-dependent stimulation of the proliferation of monkeyMBr-5 cells was found to be in the range of.0-40.0 ng/ml.

Protein Sequence MSTESMIRDV ELAEEALPKK MGGLQNSRRC LCLSLFSFLL VAGATTLFCL LNFGVIGPNK EEKFPNGLPL ISSMAQTLTL RSSSQNSSDK PVAHVVANHQ AEEQLEWLSQ RANALLANGM DLKDNQLVVP ADGLYLIYSQ VLFKGQGCPD YVLLTHTVSR FAISYQEKVS LLSAIKSPCP KDTPEGAELK PWYEPMYLGG VFQLEKGDLL SAEVNLPKYL DITESGQVYF GVIAL
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 Recombinant rat TNF-a was lyophilized from a.2 μm filtered PBS solution pH.0.
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.


Molecular function Cytokine

Methods

Chemicals and Kits

  • The biochemical tests conducted with the specific enzymatic colorimetric assay kits were all provided by, either located at Switzerland or .
  • The reagents IFCC and P-5-P were used for assay of GOT and GPT.
  • Octacalcium phosphate (OCP) and UV were used for the determination of serum calcium and phosphate ions.
  • For other determinations we used BCG for serum albumin, CHOD for cholesterol, lipase-glycerol oxidase for triglycerides; KINETIC for serum BUN, picric acid for creatinine (reaction), colorimetric oxidase for uric acid, and Sirius Red for collagen staining.
  • Doxorubicin (DR) was a product of Pfizer .
  • The kits for other determinations included SO and TBAS from Cayman (Michigan, ), the rat IL-6 EIA Kit from , and the rat TNF-α Kit from the& .
  • The sources of the antibodies used in this experiment were: PDGF Receptor β (1∶1000), phosphor-PDGF Receptor β (1∶1000) and β-actin from Cell Signaling ; phosphor-PI3K (1∶500) from Santa…
  • The biochemical tests conducted with the specific enzymatic colorimetric assay kits were all provided by, either located at Switzerland or .
  • The reagents IFCC and P-5-P were used for assay of GOT and GPT.
  • Octacalcium phosphate (OCP) and UV were used for the determination of serum calcium and phosphate ions.
  • For other determinations we used BCG for serum albumin, CHOD for cholesterol, lipase-glycerol oxidase for triglycerides; KINETIC for serum BUN, picric acid for creatinine (reaction), colorimetric oxidase for uric acid, and Sirius Red for collagen staining.
  • Doxorubicin (DR) was a product of Pfizer .
  • The kits for other determinations included SO and TBAS from Cayman (Michigan, ), the rat IL-6 EIA Kit from , and the rat TNF-α Kit from the& .
  • The sources of the antibodies used in this experiment were: PDGF Receptor β (1∶1000), phosphor-PDGF Receptor β (1∶1000) and β-actin from Cell Signaling ; phosphor-PI3K (1∶500) from Santa Cruz ; α-Smooth Muscle Actin (1∶1000) from ; CD34 (1∶400) from .
  • Chemiluminescent HRP Substrate was the product of .
  • Sodium dodecyl sulfate (SDS) and polyacrylamide gel (PAGE) were products of .

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Cytokine Expression

  • After the swimming training was completed, the rats were subjected to intraperitoneal ketamine and xylasine anesthesia and the blood samples were immediately withdrawn from the abdominal aorta.
  • The levels of IL-6 and TNF-α were measured by the at IL-6 EIA KIT provided by and the at TNF-α KIT by & according to the manufacturer’s instruction.
  • The minimal detectable limits instructed by the manufacturers for IL-6 and TNF-α are 62 and 5 pg·mL−1, respectively.

Bone marrow (BM) cell cultures

  • BM-derived cells were obtained by flushing tibiae and femurs.
  • After lysis of red blood cells with lysing solution , the remaining cells were suspended in complete medium (CM; RPMI 1640 supplemented with 10% fetal bovine serum [FBS], antibiotics, sodium pyruvate, L-glutamine, nonessential amino acids, and 2-ME; all from, , ).
  • Where indicated, the cells were stimulated with FMS-like tyrosine kinase 3 ligand (Flt3L; 200 ng/ml, , ), anti-4-1BB (5 µg/ml; clone 3H3, rat IgG2a; produced in-house), LPS (10 µg/ml), ODN-2395 (10 µg/ml), PolyI:C (10 µg/ml), TNF-α (5 ng/ml,), IFN-γ (5 ng/ml), anti-CD40 (5 µg/ml, , ), anti-μ (10 µg/ml , , ), or LA-4Ig (10 µg/ml: Bristol-Myers Squibb, , ).

Expression of markers relevant to antigen presentation and T cell activation on HBEC.

10 ng/ml TNF
  • HBEC were stimulated with either 10 ng/ml TNF (blue line), 50 ng/ml IFNg (green line), or 10 ng/ml TNF+50 ng/ml IFNg (orange line) and compared to unstimulated cells (red line).

Conditioned medium of ADSC promotes proliferation rate of cardiomyocytes.

TNF-α
  • This proliferation rate was not significantly influenced by treatment with TNF-α or IL-1β.

Culture and characterization of DCs

  • DCs were generated from rat bone marrow as previously described[6 cells/mL in medium'>RPMI-1640 medium containing 10% fetal bovine serum , 2 mM glutamine, 100 μg/mL penicillin , 100 μg/mL streptomycin , 500 U/mL of recombinant rat granulocyte-macrophage colony- stimulating factor , and 500 U/mL of recombinant rat interleukin-4 .
  • On days 2 and 4, half of the culture supernatant was removed and replaced with fresh medium containing recombinant rat granulocyte macrophage colony- stimulating factor and interleukin-4.
  • On day 6, all the loosely adherent cells were harvested, centrifuged and counted.
  • Then C6 glioma cell lysates and DCs (a ratio of 3 tumor cells to 1 DC) were incubated in fresh medium with recombinant rat granulocyte macrophage colony-stimulating factor and interleukin-4 together at 37°C in 5% CO2.
  • To stimulate DC maturation in vitro, on day 8, the cultures were additionally…
  • DCs were generated from rat bone marrow as previously described[6 cells/mL in medium'>RPMI-1640 medium containing 10% fetal bovine serum , 2 mM glutamine, 100 μg/mL penicillin , 100 μg/mL streptomycin , 500 U/mL of recombinant rat granulocyte-macrophage colony- stimulating factor , and 500 U/mL of recombinant rat interleukin-4 .
  • On days 2 and 4, half of the culture supernatant was removed and replaced with fresh medium containing recombinant rat granulocyte macrophage colony- stimulating factor and interleukin-4.
  • On day 6, all the loosely adherent cells were harvested, centrifuged and counted.
  • Then C6 glioma cell lysates and DCs (a ratio of 3 tumor cells to 1 DC) were incubated in fresh medium with recombinant rat granulocyte macrophage colony-stimulating factor and interleukin-4 together at 37°C in 5% CO2.
  • To stimulate DC maturation in vitro, on day 8, the cultures were additionally supplemented with 20 ng/mL of lipopolysaccharide and 1 μg/mL of recombinant rat tumor necrosis factor-α .
  • On day 9, cells were collected for phenotypic analysis by flow cytometric analyses and vaccination.

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