Q: Purity and validation

Key terms associated with protein purity and validation.Purity The percentage of the supplied product that is the protein of interest. Protein purity can be determined by multiple methods including SDS-PAGE, liquid chromatography (LC), and mass spectrometry (MS). The most accurate measurement of purity can be made using LC and MS together. Animal-free A protein is animal-free if has been produced without the use of any animal-derived materials. Carrier-free A protein is carrier-free if it provided to the customer without a carrier protein. Carrier proteins such as BSA are often added to proteins for stability; however, researchers often require carrier-free products for cell culture and in vivo use. Endotoxin level Endotoxins are bacterial toxins which may be present as contaminants within protein products. The LAL method is a standard method of measuring endotoxins. Endotoxin levels tend to be higher in proteins expressed using E.coli than those expressed in other systems.

Question 1

Application glossary

Accepted Answer

A list of key abbreviations for common life science applications.

Question 2

Protein products glossary

Accepted Answer

Key terms associated with our range of proteins.

Question 3

Protein product grading

Accepted Answer

Key terms and features associated with various protein product grades.

Premium grade bioactive

Cytokines that are of the highest activity and validation, perfect for preclinical cell culture and functional studies. These recombinant proteins are produced consistently to exceed our stringent thresholds.

Key features of these premium-grade proteins include:

Optimal bioactivity

Mammalian expressed

Ultra-high purity confirmed by LC/MS (>95%) and SDS-PAGE (>99%)

Ultra-low endotoxin (<0.005 EU per µg)

Size confirmed by mass spectrometry

Animal free

Carrier free

Manufactured to ISO9001

Bioactive grade

Recombinant proteins that are bioactivity validated, ideal for cell culture and functional studies.

Key features of bioactive-grade products include:

Validated bioactivity

Mammalian, E.coli, insect, yeast, or wheat germ expression

High purity (>90%) confirmed by SDS-PAGE

Endotoxin level below <1 EU per µg

Size confirmed by SDS-PAGE

Research grade

These are recombinant proteins that are non-bioactive, for use as standards within western blot, flow cytometry, and other experiments.

Key features of research-grade proteins include:

Mammalian, E.coli, insect, yeast or wheat germ expression

Purity analyzed (>80%) by SDS-PAGE

Size confirmed by SDS-PAGE

Question 4

Recombinant proteins

Accepted Answer

View all key terms associated with recombinant proteins.

Recombinant protein
A protein produced using recombinant DNA sequences acting as templates from which proteins are expressed in various expression systems via the processes of transcription and translation. When bioactivity has been confirmed they are also classed as being in a "native-like" state.

Native protein
A protein isolated from endogenous samples such as blood or animal tissue. They are in their folded and assembled form and haven’t been altered by heat, chemicals, enzyme reaction, or other denaturants.

Denatured protein
A protein that has lost its native folded structure. Denatured proteins are produced by altering the folding conditions, which can include increased temperature, altering buffer pH, or adding denaturants. Denatured proteins typically lose their biological function; however, the action of denaturation can be reversed in many cases.

Post-translational modifications (PTMs)
Modifications made to proteins after they have been synthesized, including phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis. Post-translational modifications can have many roles, including mediating proper protein folding or stability, directing a nascent protein to distinct cellular compartments, activating or inactivate catalytic activity, or otherwise influencing the biological activity of the protein.

Bioactivity
The response that a protein elicits within a biological system. The specific bioactivity of a protein is a measured using a functional assay and expressed as the ED50: the effective dose required to produce a biological response in 50% of a population. Not all proteins sold have bioactivity validated as it is not required for some applications.

Question 5

Purity and validation

Accepted Answer

Key terms associated with protein purity and validation.

Purity
The percentage of the supplied product that is the protein of interest. Protein purity can be determined by multiple methods including SDS-PAGE, liquid chromatography (LC), and mass spectrometry (MS). The most accurate measurement of purity can be made using LC and MS together.

Animal-free
A protein is animal-free if has been produced without the use of any animal-derived materials.

Carrier-free
A protein is carrier-free if it provided to the customer without a carrier protein. Carrier proteins such as BSA are often added to proteins for stability; however, researchers often require carrier-free products for cell culture and in vivo use.

Endotoxin level
Endotoxins are bacterial toxins which may be present as contaminants within protein products. The LAL method is a standard method of measuring endotoxins. Endotoxin levels tend to be higher in proteins expressed using E.coli than those expressed in other systems.

Question 6

Expression and species

Accepted Answer

Key terms surrounding protein expression and species.

Expression system
Expression systems are the biological hosts used to generate recombinant proteins. Sources used include bacteria, yeast, insect, and mammalian expression systems. Recombinant proteins can also be expressed using cell-free systems such as wheat germ extract.

HEK293/CHO
HEK293 and CHO are mammalian expression systems. As a human cell line the HEK293 system provides the full range of post-translation modifications and proper folding so can be considered superior to other systems when producing human proteins.

E.coli
E.coli is the most common protein expression system. It offers the advantage of quick production and scale-up; however, proteins produced tend to have high endotoxin levels. Mammalian proteins produced using E.coli may not have the correct post-translational modifications and they often required refolding from solubilized inclusion bodies. This means that the functional activity of the protein is often inferior when compared to insect and mammalian expression systems.

Yeast
Yeast offers similar advantages to E.coli, ie quick production, but also offers eukaryotic post-translation modifications.

Wheat germ
Wheat germ is a cell-free protein expression system; however, proteins may lack correct folding and post-translation modifications.

Baculovirus/Sf9/Sf21
Baculovirus, Sf9 and Sf21 are insect expression systems. Insect systems provide the majority of post-translational modifications found in mammalian cells and facilitate proper protein folding.

Protein tags
Protein tags are peptide sequences included within the recombinant protein construct for solubilization and purification purposes. Tags can interfere with protein activity and should ideally be removed.

All premium bioactive proteins are tag free. If a protein contains a tag, it will be listed near the bottom of the 'Description' section on its datasheet.

Species
There are many homologous proteins that perform the same function in different species. The level of similarity is determined by the degree of sequence homology. In many cases, bioactive proteins can be used across multiple species. However, they could show lower activity when compared to proteins for that specific species. For example, although a mouse cytokine could be active on human cells, a human cytokine would provide higher activity.

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