Support & FAQs

A wide range of analytical techniques can be used to measure the various properties of a protein, such as the size, charge, amino acid composition and hydrophobicity. Confirmation of the purity of growth factors by several varied techniques ensures reliability of results. When shopping for growth factors look for products where purity has been validated by multiple methods. Some common purity assessment methods are discussed below.

What is an SDS-PAGE gel and how do you interpret it? What are reducing vs non-reducing conditions?

SDS-PAGE (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) is a method that separates protein by its mass. SDS is an ionic detergent that denatures and binds to proteins to make them uniformly negatively charged. This means that when an electronic current is applied to a polyacrylamide gel, the SDS bound proteins will migrate down the gel towards the positively charged electrode, separated by size alone.

Proteins can be reduced before being run on an SDS-PAGE gel. In reducing conditions β-mercaptoethanol (β-ME or 2-ME) or dithiothreitol (DTT) is added; this reduces the disulfide bridges in proteins so that when they are run on the gel, they are better separated by size. Many of our growth factors are disulfide-linked dimers and so running non-reduced proteins on the same gel as reduced proteins allows confirmation of the correct dimeric state.

What should I look for from mass spectrometry analysis?

Mass spectrometry analysis is used to confirm the molecular mass of the intact protein and to reveal any heterogeneity that would not be evident from SDS-PAGE analysis. The resultant mass is compared with calculated mass of the protein with the assumption that all the cysteines are disulfide-linked. Multiple peaks represent different charge states of the protein.

Endotoxin levels; what problems can endotoxins cause? What level of endotoxin contamination is acceptable? 

Endotoxins are small hydrophobic lipopolysaccharide molecules, a toxic substance found in the outer cell membrane of gram-negative bacteria. Endotoxins are shed by bacteria during their cell death or when they are actively growing and dividing. Endotoxins can affect the growth or performance of cell cultures and are a major source of experimental variation. Endotoxins have a very high heat stability meaning they cannot be destroyed with regular decontamination methods such as autoclaving. Endotoxins are also hydrophobic, and consequently have a strong affinity for other hydrophobic materials such as lab plastics. Endotoxins can be avoided by cleanliness and proper lab techniques to keep endotoxin levels in a lab at bay. At Qkine, we achieve this with good aseptic technique, filter sterilization of buffers used during manufacturing processes and rigorous purification column cleaning protocols. This will minimize introduction of endotoxins into any manufacturing/research processes.

To determine the presence of endotoxins, a limulus amebocyte lysate (LAL) assay is used. This assay can detect as little as 0.01 endotoxin units (EU)/mL. One EU equals approximately 0.1 to 0.2 ng endotoxin/mL of solution. Current industry standards require endotoxin levels in growth factor lots to be less than 0.5 EU/mL, however the lower the better, and companies such as Qkine impose internal standards of <0.1 EU per µg protein.

It is very hard to determine the activity of a growth factor as they have a large range of potential effects. A luciferase reporter assay is used to quantify the response of transfected cells to a growth factor by assaying the expression of luciferase (the reporter gene). As the biological activity of a growth factor is measured by its effect on a defined cell type in standardised conditions, it may not accurately represent your own culture conditions, however it will provide confirmation that the growth factor is able to elicit a biological response.

To determine this bioactivity, a vector containing a firefly luciferase gene under the control of a growth factor appropriate regulatory region is introduced into cells, such as HEK293Ts. These are then incubated with a dilution series of the growth factor. After a defined incubation period, the bioactivity is measured by quantifying the enzymatic activity of luciferase. This involves lysing the cells and adding the luciferase substrate, luciferin. The amount of light emitted from the reaction is directly proportional to the amount of luciferase in the sample and consequently can be used as a measure of the level of response the growth factor imparts on the reporter gene. In our QC experiments, we also quantify Renilla luciferase which is expressed constitutively. This acts as a normalization control for transfection efficiency and the cell number in each well. The growth factor response can then be expressed as a firefly/Renilla ratio. Plotting concentration vs. F/R ratio allows an EC50 (half-maximal effective concentration) to be calculated.

The EC50 is the concentration of the growth factor which gives 50% of the maximum effect on the given cell population

When shopping for growth factors, you want to confirm that the protein has been established to be consistently bioactive across multiple lots, ensuring each lot you receive will be equally as active as that reported. Our quality control process includes validation with a previous lot to ensure excellent lot-to-lot consistency (e.g. Figure 1). This consistency is essential when treating cells with growth factors to prevent experiment variability. The activity of a growth factor can differ significantly depending on experimental conditions. This means that comparisons between growth factors from different suppliers are tricky to make. At Qkine, we have validated most of our growth factors against other suppliers’ proteins under our experimental conditions to ensure that we are providing you with products that are as good as, or better than, those you may have used previously.

Growth factors can be synthesised in prokaryotic cells, most commonly E. coli, or eukaryotic cells, using cell lines such as human embryonic kidney (HEK) 293 cells or Chinese hamster ovary (CHO) cells. There are benefits to each of these cell sources. Proteins produced from prokaryotic sources generally have less lot-to-lot variability and higher purity than eukaryotic systems. They have the additional benefit of being animal-free, eliminating the risks of contaminating growth factors, viruses, prions and other animal-derived ingredients.  This production method also reduces the need for foetal bovine serum (FBS), use of which is an ethical concern for many researchers. However, when it comes to complex full length mammalian proteins prokaryotic systems can fall short; protein refolding and post translational modification can differ from eukaryotic systems which can affect protein solubility and activity. For growth factors from both prokaryotic and eukaryotic sources, please be sure to check the bioactivity data before you purchase.

Innovations in growth factor production are allowing an increasing number of proteins to be produced using prokaryotic systems, and they benefit from being animal-free, while maintaining correct refolding and activity. Qkine is leading this charge with products including; TGF-β1 PLUS, the first entirely animal-free TGF-β1, and bioactive fragments of human R-spondin 1 and R-spondin 3.

We have compared our lyophilized proteins incubated at 37 °C for 3 days with those stored at -80 °C and found no difference in their bioactivity (EC50) using our standard assays. Therefore, as part of our commitment to the environment and supporting more eco-friendly packaging, we send all of our lyophilized proteins at room temperature, avoiding the need for dry ice, ice packs and gels.

Every effort is made to ensure samples are sterile; however, we recommend sterile filtering after dilution in media or the final working solution.

SDS-PAGE (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) is a method that separates protein by its mass. SDS is an ionic detergent that denatures and binds to proteins to make them uniformly negatively charged. This means that when an electronic current is applied to a polyacrylamide gel, the SDS bound proteins will migrate down the gel towards the positively charged electrode, separated by size alone.

Proteins can be reduced before being run on an SDS-PAGE gel. In reducing conditions β-mercaptoethanol (β-ME or 2-ME) or dithiothreitol (DTT) is added; this reduces the disulfide bridges in proteins so that when they are run on the gel, they are better separated by size. Many of our growth factors are disulfide-linked dimers and so running non-reduced proteins on the same gel as reduced proteins allows confirmation of the correct dimeric state.

Mass spectrometry analysis is used to confirm the molecular mass of the intact protein and to reveal any heterogeneity that would not be evident from SDS-PAGE analysis. The resultant mass is compared with calculated mass of the protein with the assumption that all the cysteines are disulfide-linked. Multiple peaks represent different charge states of the protein.

Endotoxins are small hydrophobic lipopolysaccharide molecules, a toxic substance found in the outer cell membrane of gram-negative bacteria. Endotoxins are shed by bacteria during their cell death or when they are actively growing and dividing. Endotoxins can affect the growth or performance of cell cultures and are a major source of experimental variation. Endotoxins have a very high heat stability meaning they cannot be destroyed with regular decontamination methods such as autoclaving. Endotoxins are also hydrophobic, and consequently have a strong affinity for other hydrophobic materials such as lab plastics. Endotoxins can be avoided by cleanliness and proper lab techniques to keep endotoxin levels in a lab at bay. At Qkine, we achieve this with good aseptic technique, filter sterilization of buffers used during manufacturing processes and rigorous purification column cleaning protocols. This will minimize introduction of endotoxins into any manufacturing/research processes.

To determine the presence of endotoxins, a limulus amebocyte lysate (LAL) assay is used. This assay can detect as little as 0.01 endotoxin units (EU)/mL. One EU equals approximately 0.1 to 0.2 ng endotoxin/mL of solution. Current industry standards require endotoxin levels in growth factor lots to be less than 0.5 EU/mL, however the lower the better, and companies such as Qkine impose internal standards of <0.1 EU per µg protein.

As part of our Bioactivity Guarantee, we conduct comparative quantitative bioactivity studies with current dominant suppliers to ensure the bioactivity of our proteins is equivalent or better. During our R&D process we always correct for protein concentration after reconstitution, to ensure we are comparing like-for-like and correct for any discrepancies in vial recovery.

See our technotes for available bioactivity comparisons.

Recombinant proteins can be ‘tagged’ by adding a (usually) short amino acid sequence that is not naturally occurring and can therefore be used to purify the protein from a complex mixture more easily. Other frequently used protein tags allow later protein modification such as biotinylation, coupling to other proteins for subsequent biochemical protocols or improve folding or yield during protein expression.

To improve the reproducibility and reliability of experiments relying on recombinant proteins, including biochemical assays and cell culture where these proteins are often critical components of cell culture media, it is important to use tag-free proteins unless the application specifically requires a tagged form of the protein.

For more information read our blog: Protein tags, why and why not?

Contamination with other bioactive proteins leads to a lack of inter- and intra-lab reproducibility or, perhaps most concerningly, incorrect scientific conclusions.

For more on growth factor purity read our blog: Unexpected consequences: growth factor cross-contamination in mammalian and E. coli expressed growth factors