Tumor Necrosis Factor alpha (TNF-alpha) is a key pro-inflammatory cytokine. Implicated in a host of inflammatory diseases, TNF-alpha is the target of some of the best-selling prescription medications of all time. It remains an ingredient of intense basic research efforts in multiple disciplines.
At Gene And Cell Technologies, we have developed a breakthrough manufacturing process for recombinant human TNF-alpha in E. coli. As you can see from the product characteristics below, this is one of the best characterized and purest TNF-alpha preparations available. All the while, we're able to offer it at a price that is hitherto unrivaled. It's one example of our philosophy to "Do few things, and do them well".
Product must meet the specifications shown here, or it will not be sold. As you can see in the details section below, we usually exceed them by quite a bit.
Activity: ED50 < 1 ng/ml by NFKB activation assay.
Purity: No contaminants detected by SDS-PAGE; 2 ug loading volume; Coomassie sensitive protocol with overnight destaining.
Endotoxin: < 0.01 EU / μg of protein.
Storage & Use
TNF-alpha ships as pure lyophilized powder. In this condition, it is stable for shipping at ambient temperature. Upon receipt, the powder can be stored at -20 or -80C for the long term. You may dissolve TNF-alpha in mildly alkaline buffers, such as 10 mM Tris pH 8.2. These solutions too are stable at -20C or -80C for the long term.
Avoid acidic or neutral solutions. Concentrated TNF-alpha precipitates under acidic or neutral conditions.
TNF-alpha is produced by recombinant DNA technology in E. coli. Then, it is purified using a series of FPLC and HPLC chromatography steps, including multiple affinity stages, tag removal, traditional intermediate purification steps, and polishing.
We loaded 2 ug of TNF-alpha on SDS-PAGE with reducing agent. We stained the gel with Coomassie's dye using the "sensitive protocol" with overnight destaining.
Amino Acid Sequence of TNF-alpha
We assayed TNF-alpha activity with a luciferase reporter system. In brief, we constructed a plasmid containing a minimal NFKB promoter driving firefly luciferase, as reported in the published literature (e.g. PMC4003228). We generated a HEK293T stable cell line by transfection with this plasmid. We exposed the resulting reporter cell line to dilutions of TNF-alpha as shown. After 48 hours, we removed the medium, lysed, and assayed with luciferin. We took great care to keep the time from luciferin addition to measurement brief and constant for each well. We compare 6 replica of TNF-alpha with 2 replica of our bFGF, a control cytokine known not to activate NFKB. This assay was prepared robotically.
The effective dose of half-maximal activation (ED50) is 0.65 ng/ml.
We purchased an endotoxin assay kit based on the limulus amoebocyte lysate (LAL) colorimetric method from a major US vendor. We added 1 ml of endotoxin-free water to a 100 ug vial of our TNF-alpha, and assayed for endotoxin concentration with the kit:
We have 0.72 EU/ml of endotoxin and 100 μg/ml of TNF-alpha. Therefore, we can express the endotoxin concentration as 0.0072 EU per μg of TNF-alpha protein.
We set out to discover the best conditions to dissolve recombinant TNF-alpha at and keep it in solution at high concentration. To do so, we prepared buffers at pH 6, 7 and 8, with or without the addition of 500 mM salt (denoted by the letter "S" on the vial -- for example vial "6S" means pH 6, with salt added). We added each solution to a 300 ug vial and incubated overnight.
The results can easily be assessed by eye: TNF-alpha precipitated heavily in acidic solutions, precipitated moderately in neutral solutions, and stayed soluble in alkaline solutions. The addition of salt seemed to have a mild beneficial effect in the neutral and acidic solutions. In the alkaline solution, we used light scattering spectroscopy to quantitatively assess solubility and found no difference (data not shown). Adding a high salt concentration can however cause problems during freezing and thawing. We therefore recommend to use a low-salt, or physiological salt, mildly alkaline solution for dissolving TNF-alpha.
Our favorite recipe is 10 mM Tris pH 8.2, with no further additives.