Technical Questions


Technical Questions

I just received your product and it was shipped at room temperature. Is the product okay to use?

Yes.

Lyophilized recombinant protein products are intended to be shipped at ambient temperature (20-24°C  or 68-77°F) and are not compromised when exposed to normal shipping temperatures for extended periods of time (less than 5 days). Lyophilization is a dehydration process used to increase the stability of our proteins over time and make the product more convenient for shipping by reducing packing materials and shipping expense. Our products are routinely tested for bioactivity following exposure to ambient temperatures encountered during normal shipping conditions.  The data generated over time regarding ambient shipping has demonstrated that lyophilized products, shipped at ambient temperatures, retain full activity. We will guarantee and support the performance of our products when delivered and properly stored.  If your shipment was exposed to temperatures greater than 36°C (or 96°F) for more than 3 days, please contact our technical support.

Most of our products are formulated into a stable buffer and then lyophilized for long-term storage and stability. Upon reconstitution according to the product specific instructions, it is recommended that the product be aliquoted into working volumes and stored at -20°. Adding a carrier protein such as HSA or BSA will help protect the protein and prevent the product from sticking to the wall of the vial. It is important to avoid repeated freeze thaw cycles as this can damage the protein and render the product inactive.

When I opened the vial, I didn’t see anything. How do I know there is protein in the vial?

IMPORTANT: Centrifuge vial prior to opening! Most of our products are lyophilized with a low concentration buffer and we do not add carrier proteins (BSA, HSA, sucrose, mannitol, etc.), so the few micrograms of product may not be very visible. It is recommended to spin the vial before opening to ensure the product is not near the cap, and to carefully follow the reconstitution instructions.  Follow reconsitution instructions found on the Certificate of Analysis (COA) delivered with your product.

Your datasheet and/or COA states the biological activity as an ED50. What is the Specific Activity?

The ED50 is an expression of activity at 50% of its maximum response. Specific activity is just another way of conveying the activity of the protein. To calculate a specific activity, divide 1 x 10^6 by the ED50 in ng/ml.

Specific Activity (Units/mg) = 1 x 10^6 / ED50 (ng/ml)

Is specific activity reported in units/mg the same as International Units (IU/mg)?

No.  International Units are calculated using a validated assay where the cytokine in question is tested side-by-side with an internal standard set by the WHO.  The activity of the cytokine in question is then normalized to the activity level of the standard.  

Shenandoah's cytokines are not run side-by-side with an international standard and are not normalized to international units.  We are working to include this in our quality testing.  The data reported on our documents are considered “in-house-units” and are routinely run side-by-side with competitors from the recombinant protein market, but cannot be compared to other assays directly.  Even if the same cell line is used, there are still factors that will differ from lab to lab, and comparability of results will vary greatly. 

Your Datasheet and/or COA states endotoxin in EUs/ug and I need to know ng/ug.  How do a calculate this?

       Converting endotoxin units per microgram (EU/ug) to nanograms per microgram (ng/ug) is not straightforward or exact. There are several reasons for this including the following:

  • EU is a relative term used to describe the toxic activity of endotoxin from different sources. For example, an activity of 1 EU may be equivalent to 50 pg of endotoxin from one source and 100 pg from a different source. 
  • There is 50% error inherent to the current kinetic LAL test itself.
  • Charles River, the manufacturer of the LAL test Shenandoah uses, utilizes a ratio based on a comparison of their internal standard to a government standard. This is referred to as the Reference Standard Endotoxin/Control Standard Endotoxin ratio (RSE/CSE). This ratio varies per lot of LAL test cartridges.


    Keeping in mind the aforementioned limitations, a RSE/CSE ratio of 13 EU/ng will be used as an example to calculate a general EU/ug to ng/ug conversion:

    RSE/CSE = 13 EU/ng = 0.077 ng/EU

    Shenandoah's acceptance criterion for most products is <1 EU/ug. Therefore a general conversion of our acceptance criterion for most products could be described as <0.077 ng/ug.

I want to try to do an experiment with your protein, but the bioassay you use for determining activity is not the same as my application. Will my application work with your protein?

Our products are used for many different purposes, so it would be impossible to predict every possible application. Our standard bioassay is used to confirm an accepted activity level for the product. Our proteins can be used at a broad concentration range, in many different applications, thus, it is the end user’s responsibility to determine the concentrations that work best for their specific assays.

Do most proteins show cross-species activity?

Although many human cytokines will respond nicely in mouse cell lines, and many mouse proteins will show activity on human cells, species cross-reactivity must be investigated individually for each product.

How should I store my protein?

If you have just received your protein and have not reconstituted the protein yet, it is best to store it in its current lyophilized state at -20°C.

If you have already reconstituted your protein, it is best to aliquot the protein into small volumes and store it according to the directions and temperature specified on the data sheet or Certificate of Analysis (COA) shipped with the product. To prevent loss of activity, it is recommended to follow reconsitution instructions provided on the COA and avoid repeated freeze-thaw cycles and vigorous pipetting or vortexing of the protein in solution.

Quality

I’m thinking of changing our supplier of cytokines. How do I know if your products are of equal or better quality than the competition?

All of our products go through rigorous quality control testing. Our QC testing includes the following:

  • Most endotoxin levels, as determined by kinetic LAL methods, are less than <0.01ng/ug or <0.1EU/ug of protein.
  • Most products are greater than 95% pure based on SDS-PAGE and analytical HPLC.
  • Depending on the characteristics and properties of each protein, protein content is determined by one or more of the following methods: SDS-PAGE, analytical HPLC and A280.
  • Each protein is tested for biological activity based on the protein’s known activity information. In some cases there may not be a known assay for some products, especially those that are very new to the research community.
  • We are always willing to provide a sample to any research lab that would like to compare our products side by side with the competition. If you are interested in a sample, please provide your FedEx account number for shipping with your sample request.