Protein Frequently Asked Questions

Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening the tube. Avoid multiple freeze/thaw cycles. Enzymes are particularly sensitive to freeze/thaw and may lose some activity if thawed again.
When proteins are diluted to perform an assay do not freeze and re-use the diluted protein.

If the protein is going to be used more than once, aliquot into small volumes upon first thaw, and flash freeze for long term storage. To avoid evaporation or stability issues, aliquots should not be less than 5 µl in volume. Do not thaw again more than once. Perform a quick spin before opening the tube to recover the full content of the tube.

The exact concentration of a protein is lot-specific and is indicated on the vial containing the protein.

Use formula: [µM] = [µg/mL] / MW(kDa)
Example: If you receive 200 µg of a 40 kDa protein in 100 µl, then the mass concentration is 2,000 µg/ml (2mg/ml). Therefore, your molar concentration is 2,000/40 = 50 µM (0.05 mM). Make sure to observe all the units of measurement.

Store at –80°C. Thaw on ice, spin briefly in a centrifuge so all liquid is at the bottom of the tube, aliquot ≤ 5 µl to individual single-use tubes and re-freeze immediately. Avoid repeated thaw/freeze cycles. Diluting proteins prior to storage is not recommended and may require the addition of a carrier protein such as BSA (bovine serum albumin; 0.1%-0.5%). If dilution is necessary, keep the final protein concentration above 10 µg/ml.  

The expected MW of a recombinant protein is calculated from its amino acid sequence.  Most commonly, post-translational modifications may change the apparent MW of the recombinant protein in SDS-PAGE, for example when the protein is expressed in a system that allows for glycosylation. In these cases, a broadening of the band may also be seen due to the addition of heterogeneous complex glycans in expression systems such as HEK293.

Bacterially expressed proteins have high expression rates with minimal post-translational modifications. Examples of post-translational modifications not present in bacterial recombinant proteins include glycosylation, tyrosine phosphorylation and epigenetic post-translational modifications. Despite a limited protein folding machinery, bacterial expression is preferred when proteins are properly folded and post-translational modifications are not needed. Insect cells are eukaryotic, therefore the recombinant proteins expressed in this system often have the same or very similar post-translational modifications as observed in mammalian cells.  One major difference between insect cells and mammalian is that proteins expressed in insect cells contain the simple glycan (GlcNAc)2Man9, added through N-glycosylation, whereas proteins expressed in mammalian cells contain more complex and heterogeneous glycans.

Usually, our recombinant proteins contain a tag to facilitate their detection and purification. Proteins are affinity-purified according to their tag, for example using Ni-NTA resins for a 6xHis tag, protein A columns for a Fc tag, and so forth. Gel filtration may be used to assess aggregation.  

HiP™ proteins, or High Purity Proteins, are affinity-purified recombinant proteins with a purity level >90% AND less than 10% aggregation as assessed by gel filtration. Our HiP™ trademark designates our highest standard of quality for recombinant proteins. 

His-tags are typically composed of 6–10 consecutive histidine residues at either end of the protein of interest. The protein description on our website usually indicates where the tag is located, and whether it is a 6xHis or a 10xHis. BPS commonly uses 6xHis-tag, which has a MW of 0.8 kDa, however 9xHis or 10xHis may be added as custom tags. His-tags facilitate protein detection or immunoprecipitation of the protein using an anti-His tag antibody, or purification using immobilized metal affinity chromatography (IMAC), the most common being Ni-resin. The tag's small size reduces interference with protein structure or function.

The Avi-Tag™ is an optimized 15-amino acid peptide used as substrate by biotin protein ligase BirA. It allows the highly selective addition of one biotin molecule to the lysine residue of the tag by enzymatic biotinylation. It can be added to either end of a protein and used for detection, immunoprecipitation or purification. Avi-Tag™ biotinylation is more precise than other biotinylation methods, as the biotin moiety is added only to the Avi-Tag sequence. The molecular weight of the Avi-Tag™ is 1810 Da. 

BPS uses the AviTag™ technology for most biotinylated products. Recombinant proteins are usually biotinylated in vitro using BirA ligase to covalently append biotin to the AviTag™, placed either at the N-terminal or the C-terminal end of the target. This method results in very good rates of biotinylation (typically ≥90%). To learn more, see our BirA tech note.

A PreScission™ sequence is a short linker sequence that can be inserted between a protein of interest and a tag, and is used to remove the tag from the recombinant protein when desired. This is achieved using the PreScission™ protease, also known as the human rhinovirus (HRV) 3C protease. This protease specifically cleaves the amino sequences Leu-Phe-Gln↓Gly-Pro between the Gln and Gly residues.

The Fc (fragment crystallizable) region of an antibody is the C-terminal domain responsible for interaction with cell surface Fc receptors and with proteins of the complement system. The discovery that bacterial protein A and protein G bind strongly to the Fc portion of IgGs led to the widespread adoption of purification and precipitation methods using Protein A/G resins. Recombinant proteins in which the C-terminus has been fused to the Fc region of an IgG can be detected with anti-IgG antibodies, and can be immunoprecipitated or purified with protein A/G/L resins in a straightforward fashion.

BPS Bioscience adds new proteins on a weekly basis. If you do not find your favorite protein, let us know. We are happy to design new products upon request and modify existing proteins according to your specific needs.

Recombinant proteins containing a FLAG-tag (a FLAG-tag consist of amino acids DYKDDDDK) may be affinity-purified using anti-FLAG antibody beads and eluted with free FLAG peptide. The FLAG peptide contained in the protein formulation buffer is just a left-over from the purification process. It is not due to cleavage of the tag from the protein but is residual from the elution step.
The free FLAG peptide will not interfere with most types of assays. However, if desired, it may be removed from the final formulation by monoQ upon request (a fee may be added).