Protein preparation and storage
How to store and reconstitute lyophilized growth factors
Lyophilized (freeze-dried) proteins can be considerably more stable than proteins in solution during storage. This also allows proteins to be shipped at room temperature – good for the environment and your experiments.
We thoroughly validate all storage and shipping of our proteins. In some cases, the intrinsic biochemical properties of the protein prevents effective lyophilization and reconstitution. In those circumstances, we store and ship proteins frozen in solution (on dry-ice).
All proteins should be stored in -70 °C on receipt (or -20 °C if you don’t have access to -70 °C storage).
Storage
Lyophilized store at -20°C or -80°C for up to 2 years.
Reconstituted store at -20°C or -80°C.
We recommend that single use aliquots are prepared to avoid freeze-thaw cycles.
Reconstitution instructions
- Check the resuspension solution recommendation on the product datasheet or use the quick reference calculator below. 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).
- Centrifuge briefly before reconstituting to ensure all powder is at the bottom of the vial.
- Add sufficient reconstitution solution to give a protein concentration of 0.05–1.0 mg/ml. For maximum protein recovery of larger sample sizes, reconstitute in approximately 80% of the total volume, leave to stand for 1 min and then transfer the reconstituted sample to a fresh tube and rinse the original tube with the remaining solution, pool the samples.
- Allow the reconstituted sample to stand at room temperature 1 min before gently agitating, do not vortex, foaming must be avoided
- Prepare single-use or stock aliquots. This stage will depend on your final application so please adapt as appropriate. All our proteins are supplied carrier protein-free. If compatible with your work and you are storing at lower concentrations (<50 ug/ml) adding carrier protein is highly advised (usually 1% w/v high purity BSA or equivalent – ensure all buffers are sterile-filtered).
- Prepare single-use aliquots whenever possible to avoid freeze-thaw cycles which can damage the proteins and reduce bioactivity. Store aliquots at -70 °C (or -20 °C)
Reconstitution solution finder
Reconstitution calculator
Desired concentration (ug/ml)
Volume (ul)
How to store and prepare growth factors shipped in solution
Some growth factors are most stable stored and shipped frozen in solution. Our growth factors are highly pure and don’t contain any carrier proteins so you can add these if they are compatible with your work and you need to prepare single-use working aliquots with protein concentrations <50 μg /ml.
All proteins should be stored in -70 °C on receipt (or -20 °C if you don’t have access to -70 °C storage). Never store protein aliquots at 4°C.
Storage
Store at -20°C or -80°C. We recommend that single use aliquots are prepared to avoid freeze-thaw cycles.
Handling instructions
- Thaw the vial on ice, spin briefly
- Prepare single-use or stock aliquots. This stage will depend on your final application so please adapt as appropriate. All our proteins are supplied carrier protein-free. If compatible with your work and you are storing at lower concentrations (<50 ug/ml) adding carrier protein is highly advised (usually 1% w/v high purity BSA or equivalent – ensure all buffers are sterile-filtered).
- Prepare single-use aliquots whenever possible to avoid freeze-thaw cycles which can damage the proteins and reduce bioactivity. Store aliquots at -70 °C (or -20 °C)
- We recommend sterile filtering after dilution in media or the final working solution
FAQs
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 2 years (either at -20 oC or -80 oC).
Once reconstituted, recombinant proteins can be stored at 4oC 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 2 years. However, on occasion lyophilization can cause partial loss of activity and damage protein structure, so some proteins are supplied in buffer.
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.
TGF beta family proteins and other growth factors can be very poorly soluble in physiological solutions. Please follow the handling guidance for lyophilized cytokines below to minimize loss of protein due to precipitation or adsorption to plastic. We advise storing the recombinant protein at very low pH before dilution in cell culture media or final working solutions. Low pH will also assist in maintaining the correct disulfide structure of the protein by minimizing disulfide bond exchange reactions.
- Resuspension in physiological buffers may cause precipitation of stock solutions, hence we recommend dissolving our lyophilized cytokines in 10 mM HCl (1:1000 dilution of concentrated HCl) while keeping the protein concentration at 50 µg/ml or above, in order to avoid loss by adsorption to plasticware.
- To ensure you recover all of the protein, let the sample sit for a few minutes with the solubilization buffer at room temperature and pipette gently up and down (avoid foaming).
- Rinse the tube with some more 10 mM HCl and pool with the rest.
- The protein is tolerant of some freeze and thaw cycles, but as always with proteins, it is better to aliquot and store frozen.
- Our proteins are supplied carrier-protein free. If compatible with your work, add carrier protein of your choice such as BSA, HSA or gelatin to further minimize loss by adsorption.
- Store in -80°C for long term storage. -20°C for short-term.
Every effort is made to ensure samples are sterile; however, we recommend sterile filtering after dilution in media or the final working solution.
