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FAQs

 

Protein FAQs

 

What is a carrier protein?

A carrier protein is an additional protein added to the lyophilized recombinant protein during reconstitution to increase the stability of the diluted recombinant protein during storage, freezing, and use.
 

What is an acceptable carrier protein for recombinant protein reconstitution?

Bovine Serum Albumin (BSA) is the most common carrier protein used for recombinant protein reconstitution. However, if working with bovine recombinant protein, use a non-bovine carrier protein such as Equine Serum Albumin (ESA). 
 
If using the recombinant protein for in vitro cell culture stimulation, it is often useful to reconstitute the recombinant protein directly in the complete cell culture medium containing serum or other acceptable carrier protein instead of a BSA/ESA solution.
 

What concentration of carrier protein is required for reconstitution?

The concentration of carrier protein should be at least 0.1% in the final solution. Additional carrier protein can be used, if desired. Typically, a 0.1-10% BSA solution in 1X PBS is an acceptable reconstitution solution.
 

What is an acceptable volume for recombinant protein reconstitution?

Typically, 100 µL is an acceptable reconstitution volume for our 5 µg and 25 µg vials of recombinant protein. Our 5 µg and 25 µg vials have a maximum volume of 500 µL. 
 
Typically, 1 mL is an acceptable reconstitution volume for our 100 µg vials of recombinant protein. Our 100 µg vials have a maximum volume of 2 mL.
 

What concentration is the recombinant protein upon reconstitution?

The concentration of recombinant protein is dependent on the reconstitution volume. For example, if reconstituting 5 µg of recombinant protein in 100 µL carrier protein solution, the concentration is 5 µg/100 µL or 50 µg/mL. 
 

What is the biological activity of the recombinant protein?

The biological activity of the recombinant protein is listed on the Data Sheet. If the biological activity is not listed on the Data Sheet, we are in process of testing the recombinant protein bioactivity. Species-specific bioassays are not typically available for the majority of our proteins. Thus, these bioassays need to be developed. We choose to make our recombinant proteins available during this testing period. 
 

What are the International Units of bioactivity for the recombinant protein?

We only report our recombinant protein bioactivity in units of mass (i.e. µg), not in units of bioactivity (i.e. International Units). International Units of bioactivity are the amount of product required to generate a specific measurement of bioactivity in a specific assay under specific conditions. International Units of bioactivity are protein and assay dependent. In addition, typically only human recombinant proteins have these specific bioassays developed that correlate to International Units of bioactivity. International Units of bioactivity measurements are usually not clearly defined for non-human species. Given that bioassays typically produce highly variable results, the determination of International Units of bioactivity is also highly variable. Mass, however, is not variable, so it provides a more accurate measurement. Thus, Kingfisher Biotech reports all recombinant protein bioassay results in units of mass.

What are the advantages of yeast as an expression system, and how does it compare to an E. coli expression system?

Yeast has several advantages as an expression system, and the recombinant proteins generated by yeast have several characteristics that make them ideal for many applications including cell culture. The yeast expression system used by Kingfisher Biotech secretes recombinant protein into the media. This secretion is typically considered the first step in the purification process because yeast cells inherently secrete very few native proteins. Recombinant proteins secreted by yeast are processed by a Golgi apparatus, thus they may be post-translationally modified. These modifications can include glycosylation, phosphorylation and sulfation. There are many proteins that require modifications for protein function, proper folding or solubility. Another added benefit of a yeast expression system is that yeast do not have a bacterial cell wall like E. coli, thus there is no endotoxin (lipopolysaccharide) present. Yeast are naturally endotoxin-free.

E. coli-expressed recombinant proteins do not contain post-translational modifications (E. coli do not contain a Golgi apparatus). Recombinant proteins made in E. coli are often localized to inclusion bodies. Purification from these inclusion bodies can require harsh conditions to free the recombinant protein, followed by subsequent refolding processes. These harsh treatments can affect the function of the protein.
 

ELISA FAQs

How many assays can I perform with the Do-It-Yourself ELISA?

The Do-It-Yourself ELISA kits are the stand-alone unconjugated antibody (capture antibody), recombinant protein (standard), and biotinylated antibody (detection antibody). Each of these components can be bought individually, if desired (or as components run out). These antibodies have been shown to function together with the standard provided in an ELISA. Evaluating the individual components’ Data Sheet will give a representative working concentration to help determine the number of assays that can be performed. Typically, the capture antibody is the limiting reagent.
 
For example, the Swine IFNα1 Do It Yourself ELISA (DYI0724S-003) includes:
 
100 µg of Anti-Swine IFNα1 Polyclonal Antibody (PB0445S-100):
The recommended titer for this reagent in ELISA is 1-5 µg/ml. If coating with 100 µl per well in a 96-well plate, this will require approximately 10 ml of reagent to coat a 96-well plate. Thus, 10 µg-50 µg per plate is required, depending on titer used. That means the reagent provided should coat 2-10 plates.
 
5 µg of Recombinant Swine IFNα1 (RP0010S-005):
The optimal standard concentrations will need to be determined by the user. If using a top standard concentration of 50 ng/ml (0.05 µg/ml), assuming 0.5 ml of standard is required per plate, this should make enough standard for 200 assays.
 
50 µg of Biotinylated Anti-Swine IFNα1 Polyclonal Antibody (PBB0451S-050):
The recommended titer for this reagent in ELISA is 0.1-0.5 µg/ml. If detecting with 
100 µl per well in a 96-well plate, this will require approximately 10 ml of reagent to detect a 96-well plate. Thus, 1-5 µg per ELISA is required, depending on titer used. That means the reagent provided should detect in 10-50 assays.
 
Thus, depending on optimization results, there can be quite a large difference in the number of ELISAs able to be performed with the provided components. As mentioned before, each of these components can be purchased individually. Most likely, the capture antibody will need to be purchased individually as that reagent will be depleted first before the other components (it typically is the limiting reagent).
 

The VetSet ELISA Kits are validated for cell culture supernatants. Can we use other sample types in the assay, such as serum or plasma?

Many of our customers use our VetSets for serum, plasma, or other sample type detection. In order to use a different sample type, validation of the Standard and Sample Diluent in the assay is required. The optimal diluent should provide linear results (i.e. when the sample is diluted, similar values are obtained when correcting for the dilution factor) and have 80-120% recovery (i.e. if a known quantity of standard is added to a sample type, 80-120% of what was added should be measured in the assay).
 
In order to verify if the sample diluent works with a desired sample type, a linearity and recovery assay will need to be performed. This assay is simple to perform. Simply prepare a standard curve like normal using your test sample diluent. Add/spike a known quantity of standard into a test sample. Add/spike the same quantity of standard to the test sample diluent (this is the 100% recovery value). Be sure to run a non-standard spiked test sample in the assay to subtract out any background values from the test sample. Prepare multiple serial dilutions of the diluent standard-spiked sample, the test standard-spiked sample, and the non-standard spiked test sample with the test diluent (i.e. 1:2, 1:4, 1:8, 1:16, 1:32, etc.). Optimal recovery is when the standard spiked sample value (subtract out non-standard spiked sample background) is 80-120% of the diluent standard spiked sample result. In addition, the results should give similar values when corrected for dilution (linearity). 
 
Often, a 1:4 to 1:8 dilution with 4% BSA in DPBS (Reagent Diluent) is sufficient to achieve linearity of the sample and obtain optimal recovery. If optimal results are not obtained with 4% BSA in DPBS, a higher sample dilution or a different sample diluent will need to be used. Other diluents to try are 10-50% serum (such as fetal bovine serum or equine serum) in DPBS (as long as the serum does not contain the protein of interest that could cross-react with the ELISA). Additionally, increasing the BSA content in DPBS buffer may also increase linearity and recovery.
 

 

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