Recombinant Human TNF alpha Protein

What it means: This TNF-α is produced in Human cells rather than in E. coli or insect cells. The manufacturer describes the source as human cells and does not name a specific cell line.

Why it matters:

• Production in a human host avoids bacterial host-cell contaminants that can matter in sensitive primary-cell, immune-cell and stem-cell cultures.
• Human-cell expression supports mammalian folding and processing; the manufacturer describes this protein as a glycosylated homotrimer.
• Together with the animal-free, xeno-free manufacturing format, it suits fully human culture systems.

How to interpret here: An expression system of Human cells means the protein was made and processed in human cells. It is intended for inflammation and immune-cell research with human cells.

Tip: When switching from a TNF-α made in another host, re-titrate the working concentration rather than assuming the same activity per microgram.

What it means: The product is Tag-free: no His, Fc, GST or other affinity tag is fused to the TNF-α sequence, and no tag needs to be removed before use.

Why it matters:

• A tag adds extra amino acids that can change folding, receptor binding, stability or the apparent molecular weight of a small signaling protein.
• In defined culture media every added component is a variable; a tag-free protein keeps the ligand as close as possible to the native sequence.
• Tag-free proteins are also preferred when results will be compared with published protocols that used untagged cytokines or growth factors.

How to interpret here: Tag-free here means the vial contains only TNF-α protein. It cannot be captured or detected with anti-His, anti-GST or Protein A reagents, so plan detection with an antibody against TNF-α itself.

What it means: Purity of >95% by SDS-PAGE means that when the product is run on a denaturing gel and stained, at least 95% of the stained protein is TNF-α. It is the purity specification the manufacturer gives for this product.

Why it matters:

• High purity limits other proteins in the vial that could act on cells or interfere with downstream readouts.
• Dosing is done by protein mass, so a pure preparation makes the amount of active protein added more accurate and comparable between experiments.
• Purity is specified together with a low endotoxin level of <0.5 EU/µg.

How to interpret here: A specification of >95% by SDS-PAGE is a minimum value. The exact result for each lot is on the Certificate of Analysis (CoA), available from BioHippo on request.

Tip: SDS-PAGE purity shows how clean the preparation is, not how active it is; check the activity data separately.

What it means: The species is Human: the amino-acid sequence is that of human TNF-α. The protein is also produced in human cells, so both the sequence and the expression host are human.

Why it matters:

• Matching the species of the protein to the species of the cells avoids differences in receptor affinity between species.
• Human sequence is the usual choice for work with human primary cells, stem cells and human cell lines.
• Recording the species in protocols matters because the same protein is sold from several species by many suppliers.

How to interpret here: Human here refers to the protein sequence. When reproducing a published protocol, check that the protocol also used human TNF-α; if it used a protein from another species, the working concentration may need adjusting.

What it means: The product is supplied Lyophilized, that is freeze-dried to a powder, from a 0.2 µm-filtered PBS solution and without carrier protein.

Why it matters:

• Lyophilized protein is more stable during storage than a solution, which gives a long shelf life when stored correctly.
• The stock concentration is set at reconstitution, so it can be matched to the volumes used in the protocol.
• As supplied, store at −20°C to −80°C for up to 12 months from receipt; after reconstitution keep it under sterile conditions at 2°C to 8°C for up to 1 month or at −20°C to −80°C for up to 3 months.

How to interpret here: Lyophilized means the vial must be reconstituted before use: briefly centrifuge the vial before opening, then reconstitute in sterile PBS containing 0.1% endotoxin-free recombinant human serum albumin.

Tip: Avoid repeated freeze–thaw cycles of the reconstituted protein; a manual defrost freezer is recommended.

What it means: The manufacturer lists the molecular weight as 17 kDa. Separately, the manufacturer describes the protein structure as a glycosylated homotrimer.

Why it matters:

• Molecular weight is the first check of identity when the protein is run on a gel or analysed by other methods.
• The manufacturer gives a single value rather than a range; it is quoted here exactly as stated.
• Gel conditions, such as reducing versus non-reducing sample buffer, can change the observed band, so results should be compared only between gels run under the same conditions.

How to interpret here: 17 kDa is the value given by the manufacturer. Refer to the lot-specific Certificate of Analysis (CoA), available from BioHippo on request, for the data that apply to each lot; the CoA is the reference for identity and purity checks on a specific lot.

Tip: Record the gel conditions and molecular-weight marker used whenever a band is compared with this value.

What it means: Biological activity is tested by the cytotoxic effect of TNF-α on L-929 cells in the presence of actinomycin D. In short, the assay is L-929 cytotoxicity with actinomycin D.

Why it matters:

• A cell-based test confirms that the protein is functionally active, which purity alone cannot show.
• Induction of cell death is one of the defining activities of TNF-α, so a cytotoxicity assay checks the function most relevant to cell-death studies.
• Results from the same assay type can be used to compare lots, or to compare this product with other preparations tested in a similar way.

How to interpret here: L-929 cytotoxicity with actinomycin D confirms that the TNF-α is active in a responsive cell-based test. It is not a recommended working dose; the concentration for each experiment depends on cell type, medium and readout and should be optimized for each protocol.