Recombinant Human BMP-4 Protein

What it means: This BMP-4 is produced in HEK293 human cells, a human embryonic kidney cell line widely used to make secreted human proteins, rather than in bacteria or insect cells.

Why it matters:

• BMP-4 is a disulfide-linked homodimer; expression in HEK293 supports mammalian protein folding and post-translational processing of the dimer.
• A human host avoids bacterial host-cell contaminants that can affect sensitive stem-cell and developmental cultures.
• The manufacturer makes the protein in an animal-free, xeno-free format, which suits fully human culture systems.

How to interpret here: An expression system of HEK293 means the ligand was made and secreted by human cells. This is consistent with its main use in human iPSC and ESC differentiation, mesoderm induction and organoid work.

Tip: When switching from a BMP-4 made in another host, re-titrate the working dose rather than assuming the same activity per microgram.

What it means: The protein is Tag-free: no His, Fc, GST or other affinity tag is fused to the BMP-4 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 signaling protein.
• In defined stem-cell and organoid cultures every added component is a variable; a tag-free protein keeps the ligand close to the native sequence.
• Tag-free proteins are also easier to compare with published protocols that used untagged factors.

How to interpret here: Tag-free here means the vial contains only the BMP-4 protein. It cannot be captured or detected with anti-His, anti-GST or Protein A reagents, so plan detection with an antibody against BMP-4 itself.

What it means: Purity of >95% by SDS-PAGE means that when the protein is run on a denaturing gel and stained, at least 95% of the stained protein is the BMP-4 band.

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 ligand added more accurate and comparable between experiments.
• Purity is checked together with endotoxin (<0.5 EU/µg) and biological activity before each lot is released.

How to interpret here: A specification of >95% by SDS-PAGE is a minimum value. The exact result for the lot received 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 BMP-4 (bone morphogenetic protein 4). 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 factor to the species of the cells avoids differences in receptor affinity between species.
• Human sequence is the usual choice for human iPSC, ESC, organoid and primary human-cell work.
• 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 it also used Human BMP-4; if it used a protein from another species, the working concentration may need adjusting.

What it means: The protein is supplied Lyophilized (freeze-dried) 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 chosen at reconstitution, so it can be matched to the volumes used in each protocol.
• As supplied, store at −20°C to −80°C for up to 12 months from receipt; after reconstitution keep it at 2°C to 8°C for up to 1 month or at −20°C to −80°C for up to 3 months under sterile conditions.

How to interpret here: Lyophilized means the vial must be reconstituted before use: briefly centrifuge the vial before opening, then reconstitute in sterile 4 mM HCl containing at least 0.1% human serum albumin or bovine serum albumin, mix gently and prepare single-use aliquots.

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

What it means: The endotoxin specification is <0.5 EU/µg: less than 0.5 endotoxin units per microgram of BMP-4 protein. Endotoxin (bacterial lipopolysaccharide) is a common contaminant of recombinant proteins.

Why it matters:

• Endotoxin can trigger inflammatory signaling and change the behavior of stem cells, progenitors and immune cells, even at low levels.
• In differentiation workflows an unwanted inflammatory signal can shift lineage outcomes and add variability between lots.
• Endotoxin is checked on each lot together with purity and biological activity before release.

How to interpret here: A value of <0.5 EU/µg is a maximum. The amount of endotoxin added to a culture scales with the amount of protein used, so higher working doses add proportionally more; lot release data are on the Certificate of Analysis.

Tip: The manufacturer does not give a fixed molecular weight for this product and refers to the lot-specific CoA and SDS-PAGE profile.

What it means: Activity is checked in ATDC-5 mouse chondrogenic cells by measuring alkaline phosphatase induction. A representative assay gave EC50 approximately 5 ng/mL, the concentration that produces half of the maximal response.

Why it matters:

• A cell-based activity test confirms that the protein is functionally active, which purity alone cannot show.
• Alkaline phosphatase induction in a chondrogenic line is a direct readout of BMP receptor signaling.
• Activity is checked on each lot together with purity and endotoxin before release.

How to interpret here: EC50 approximately 5 ng/mL describes potency in the ATDC-5 assay for a representative lot. It is not a recommended working dose; concentrations for stem-cell differentiation depend on cell line, medium, stage and exposure time.

Tip: Confirm the working dose with a dose-response study in the target cells and protocol.