3C Protease

What it means: Proteases is the enzyme class this product belongs to. The class states what chemistry to expect — the substrate acted on, the bond made or broken, and the buffer, pH and cofactor conditions the reaction needs. Here the working function is that 3C Protease recognises the LEVLFQ↓GP sequence and removes fusion tags with high specificity.

Why it matters:

• Products in the same class (Proteases) are far more likely to be interchangeable in a protocol than two enzymes that merely share a molecular weight or an expression host.
• Reaction conditions optimised for one member of a class do not always transfer unchanged to another member, so read the class together with the unit definition and buffer composition.
• The typical catalogue application for this class is removing affinity tags from recombinant fusion proteins during purification.

How to interpret here: This preparation is supplied for removing affinity tags from recombinant fusion proteins during purification; use the Proteases classification as the first filter when looking for a comparable product, then narrow by unit definition.

Tip: Filter the catalogue by Proteases first, then by unit definition — it is faster than searching by product name.

What it means: Source is the organism the enzyme sequence originates from. For this product the source is HRV-14.

Why it matters:

• Sequence, post-translational modification, thermal stability and the optimal reaction window all track with the source organism, so two enzymes catalysing the same reaction from different sources can behave differently at the bench.
• The protein is produced recombinantly in E. coli rather than purified from native tissue, which gives better lot-to-lot consistency and avoids co-purifying incidental activities from the original organism.
• Activity units defined against one supplier's enzyme do not always convert directly to another's, even at the same source.

How to interpret here: Read HRV-14 together with the unit definition and buffer composition when transferring a published protocol to this material.

Tip: Record the source and the lot number on your assay sheet so results stay traceable when you re-order.

What it means: Expression system is the host used to produce the recombinant protein. This enzyme is expressed in E. coli.

Why it matters:

• The host determines folding, disulfide formation, glycosylation where relevant, and above all the host-cell impurity profile in the final vial.
• When an assay is sensitive to host background — trace nuclease or protease carryover ahead of a nucleic-acid or cleavage readout — match the expression system to the assay rather than assuming every recombinant preparation behaves alike.
• An enzyme that requires eukaryotic modification will not reach full specific activity from a prokaryotic host, so check reported activity rather than sequence alone.

How to interpret here: E. coli expression here is followed by purification to >95% as determined by sds-page; if your workflow needs a different host, a custom preparation can usually be arranged.

Tip: Run a no-enzyme control on first use to establish the background contributed by buffer and host.

What it means: Purity states the proportion of total protein migrating as the expected band. This product is supplied at >95% as determined by SDS-PAGE.

Why it matters:

• Purity is assessed by SDS-PAGE, so it is a statement about composition, not about catalytic performance.
• A very pure preparation with low specific activity and a less pure one with high specific activity can give an identical result in the same reaction — always read purity alongside the unit definition.
• Purity also sets how much total protein enters the reaction, which matters when downstream mass spectrometry or gel analysis has to resolve the product from the enzyme.

How to interpret here: This lot is specified at >95% as determined by SDS-PAGE; for applications where a contaminating activity would confound the readout — tag cleavage immediately before a nuclease assay is the classic case — include a control digest without substrate.

Tip: Check the SDS-PAGE image on this page to see the band pattern for yourself before scaling up.

What it means: Form is the physical presentation of the material as shipped. This product is supplied in Liquid form.

Why it matters:

• The Liquid presentation determines how to aliquot, thaw and store the enzyme after it arrives.
• Repeated freeze-thaw cycles are the most common cause of activity loss, so prepare single-use aliquots at first thaw instead of returning the stock vial to the freezer.
• Buffer components carried over from the formulation — glycerol, salt or a reducing agent — can affect electrophoresis, dialysis or mass spectrometry, so account for the dilution factor.

How to interpret here: Formulation: 50mM Tris-HCl, pH 8.0, 150mM NaCl, 10mM EDTA, 1mM DTT, 50% glycerol Storage: Use a manual defrost freezer and avoid repeated freeze thaw cycles. Store at 2 to 8°C for frequent use. Store at -20 to -80°C for twelve months from the date of receipt.

Tip: Label each aliquot with the lot number so any change in performance can be traced back to a specific vial.

What it means: Predicted molecular weight is calculated from the expressed amino acid sequence, including any purification tag. For 3C Protease it is 47.46 kDa.

Why it matters:

• It is a calculated value, not a measured mass — which is why it is labelled predicted rather than observed.
• The band on a gel can differ for ordinary reasons: modifications add mass, highly charged or proline-rich sequences migrate anomalously, and incomplete reduction shifts a band upward.
• The predicted mass is what you need to convert between mass and molar amounts when setting an enzyme-to-substrate ratio.

How to interpret here: Treat 47.46 kDa as an identity check rather than a specification, and compare it against the SDS-PAGE image supplied with the product before concluding that a lot is wrong.

Tip: Note the tag status when comparing this value with a published mass for the native enzyme.