Support & FAQs

Cells cultured with DISC™ have been used in a variety of downstream applications, including FACS, RNA/DNA extraction, nucleofection, differentiation, organoid generation, and others with no negative effects. If necessary for differentiation protocols, DISCs™ can be cleanly removed using a low powered vacuum tip.

The protein concentration can be adjusted based on the number of DISCs™  added and the medium volume. For example, placing one FGF-2 DISC™  into 2 ml of media will release FGF-2 at 10 ng/ml. Adding an FGF-2 DISC™  to 4 ml of medium will half the release to 5 ng/ml. Similarly, adding 2 FGF-2 DISCs™  to 2 ml of medium will double the release to 20 ng/ml.

Cultures should be monitored, and additional medium changes performed as needed, particularly for high density cultures that may require more frequent medium changes i.e. 3x rather than 2x a week. Additional buffers, such as sodium bicarbonate or HEPES, are rarely needed to maintain pH levels in very dense and metabolically active cultures.

IGF-1 LR3 has an arginine instead of a glutamic acid at the third position in its amino acid sequence and an additional 13 amino acids at its N-terminus. IGF-1 LR3 remains an agonist of the IGF-1 receptor, but it has very low affinity for IGFBPs, which inhibit IGF-1, making it more potent. IGF-1 LR3 also has a significantly longer half-life than IGF-1.

FRS Pioneer may be a good fit if you:

• culture commonly used immortalized cell lines
• want to reduce batch-to-batch variability associated with fetal bovine serum
• prefer chemically defined, animal-free media components
• use standard basal media and routine culture workflows
• are comfortable handling cell adhesion separately (or are comfortable retaining 1% FBS to facilitate adhesion)
• value reproducibility and experimental control
• wish to substantially reduce FBS usage for primary or organoid cell culture systems

FRS Pioneer may not be the right fit if you:

• need high-density or cell type-specific optimized cell culture media
• want a complete FBS replacement for a specific primary cell type without additional optimization

Adding a carrier protein increases protein stability, extends shelf life, allows the protein to be stored at more dilute concentrations, and helps avoid the product sticking to the walls of the vial. Consequently, adding carrier proteins can be very helpful. However, most carrier proteins are animal-derived so for animal-free and sensitive applications and assays for which BSA might interfere with the assay, carrier-free proteins are the way to go. If compatible with your work, you can add your own carrier proteins, such as BSA, HSA or gelatin when you make aliquots. Adding your own carrier allows you to select an application-compatible carrier. We recommend always using a high-purity carrier protein.

Growth factor shelf life is influenced by many factors, including storage state, buffer ingredients, storage temperature and the stability of the protein itself. Generally speaking, growth factors are most stable when lyophilized. Our lyophilized proteins have been verified to be stable at room temperature for 3 days, or frozen for up to 1 year (either at -20 ^oC or -80 ^oC).

Once reconstituted, recombinant proteins can be stored at 4^oC for 1-2 weeks, however we recommend freezing single use aliquots and defrosting fresh each time to ensure maximum protein stability and activity. Reconstituted proteins can be stored frozen for up to 1 year. When freezing reconstituted growth factors prepare single use aliquots. Every freeze-thaw cycle may cause some denaturation of the protein so repeated freeze-thaw cycles should be avoided. Always check the product sheet for freezing recommendations, but generally -20°C is suitable for short-term storage and -80°C for long term storage.

Lyophilized (freeze dried) proteins are considerably more stable than proteins in solution, consequently many proteins ship in lyophilized forms. This extends the time and temperature range at which the proteins can be shipped while maintaining the activity of the protein – good for the environment and your experiments. Lyophilized proteins are generally stable at room temperature for one month (we verify stability over a standard 3-day shipping cycle), or frozen at -20 ^oC or -80 ^oC for 1 year. However, on occasion lyophilization can cause partial loss of activity and damage protein structure, so some proteins are supplied in buffer.

As part of our standard QC we test every lyophilized protein 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. We have also tested a range of proteins for 14 days at room temperature and with multiple freeze-thaw cycles to confirm they retain their bioactivity (see application note).

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.

Address: 1 Murdoch House, Garlic Row, Cambridge, CB5 8HW, UK
Tel No: +44 (0) 1223 491486

Company registration no: 10470662
VAT No: GB265464484

We accept the following currencies via our online store:

• GBP: Pound sterling (£)
• EUR: Euro (€)
• USD: United States (US) dollar (US$)

To change the currency displayed on our website, simply click ‘currency’ on the top navigation bar and select your local or preferred currency from the dropdown list.

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.

Please order online (where you can upload a PO), email [email protected] or phone +44 (0) 1223 491486 / USA toll free 1-866 877 2185.

Our team are always happy to help and if you need reserved stock from batches you have validated, bulk orders or any further information, please contact us by phone or email ([email protected])

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.

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.

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?

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.

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

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

Qkine has implemented an ISO9001:2015 compliant quality management system encompassing its entire R&D, manufacturing, and operations functions. Embedding this internationally recognized standard demonstrates a commitment to consistently meet and exceed customer expectations and applicable statutory and regulatory requirements. UKAS accredited BSI, respected globally, was selected to certify the quality management system giving enhanced confidence in the assessment of best practice.

ISO9001: 2015 Certificate number: FS 747253

Location: 1 Murdoch House, Garlic Row, Cambridge, CB5 8HW, UK

Qkine Quality Policy

The EMA/410/01-Rev3 regulation is not directly applicable to our products as it only applies to medicinal products and the use of animal-derived products as raw materials, which we do not use in any of our manufacturing processes. However, for peace of mind, we are in the process of confirming that everything we use both directly and indirectly for manufacturing our products is compliant with the regulation.

• Lyophilized store at -20°C or -80°C for up to 1 year
• Reconstituted store at -20 °C or -80 °C
• We recommend that single use aliquots are prepared to avoid freeze-thaw cycles

1 – Check the resuspension solution recommendation on the product datasheet or use the quick reconstitution solution finder above*.

2 – Centrifuge briefly before reconstituting to ensure all powder is at the bottom of the vial.

3 – Add sufficient reconstitution solution to give a protein concentration of 0.05–1.0 mg/ml.

4 – Allow the reconstituted sample to stand at room temperature for >1 minute before gently agitating, do not vortex and avoid foaming.

5 – Prepare single-use aliquots whenever possible to avoid freeze-thaw cycles which can damage the proteins and reduce bioactivity. Store aliquots at -80 °C (or -20 °C)**.

NOTES

*Physiological buffers may cause precipitation of proteins during reconstitution. This is especially important for TGF beta family and other related growth factors so it is very important that these are reconstituted in Solution A (10 mM HCl, 1:1000 dilution of concentrated HCl).

**All our proteins are supplied carrier protein-free. If compatible with your work and you are storing at lower concentrations (<50 μg/ml) adding carrier protein is highly advised (usually 1% w/v high purity BSA or equivalent – ensure all buffers are sterile-filtered).

StemBeads® can be easily removed prior to analysis by washing 1-2 times with PBS, DMEM, F12 or any other basal medium preferred. For suspension cultures, there are a variety of strainer pore sizes to allow for separation. Cells cultured with StemBeads® have been used in a variety of downstream applications, including but not limited to: immunofluorescence, FACS, RNA/DNA extraction, nucleofection, differentiation etc.

The protein concentration can be adjusted based on the number of StemBeads® that you use. The release rate has a linear correlation with the amount of StemBeads® used. For example, 8 µl of StemBeads® per ml of media releases at a concentration of 10 ng/ml. However, if you desire half of that concentration, such as 5 ng/ml, then adjust the concentration to 4 µl of StemBeads® per ml media to achieve the concentration of 5 ng/ml.

With growth factor levels stabilized, spontaneous differentiation is reduced, pluripotency is increased, and feeding schedules are reduced to only 2-3 times per week. StemBeads are added to your medium to achieve a desired growth factor release, sustained over several days. Following, the beads are washed from the culture, the medium is replaced, and the process repeated as required.

No, this is not a problem or indication of cell quality. Extensive testing has been performed to access the cell quality when using StemBeads®. All phenotypic and functional assays have shown cell quality as good or better than current feeding regimens. For best assessment of cells, wash cells once prior to observation.