1. How to determine the quantity of proteins and cytokines?
Normally we determine the concentration of proteins and cytokines by measuring their UV absorbance. The concentration of proteins and cytokines is calculated by their extinction coefficients. We also use SDS-PAGE, HPLC and other methods to quantify the proteins and cytokines.
Before releasing the recombinant proteins and cytokines for sale, they are thoroughly analyzed to ensure the following:
1. Authenticity: by N-terminal sequence analysis and, when possible, by SDS-PAGE, RP-HPLC, and Mass Spectrometry analyses versus standards.
2. Purity: by SDS-PAGE and RP-HPLC analysis
3. Biological Activity: by the relevant in vitro or in vivo bioassay.
4. Protein content: by UV spectroscopy, SDS-PAGE analysis and, when possible, by HPLC analysis versus the calibrated standard solution.
5. Endotoxin contamination: by kinetic LAL method.
6. Microbiological contamination: by membrane filtration method; protein solutions are sterile-filtered prior to vialing
2. Why is the quantification of the protein generated by my assay different from the one on the label?
Different assays generate different quantification result. Sometimes the discrepancy can be significant if you conduct a different assay. It is also possible that the protein forms aggregations during storage which causes loss after reconstitution and centrifugation. We run quality control tests for each batch of proteins and cytokines. However, it is still inevitable that a few vials are not the same with the rest of the same batch. This happens very rare and is minimized by our effort on quality control.
3. Why are the proteins lyophilized? How will lyophilization affect the characteristics of the proteins?
Lyophilization (freeze-drying) is a technology applied to increase the stability and shelf life of proteins, and to decrease shipping cost. Lyophilization may cause loss of activity or aggregation of proteins. We take steps to ensure proper control, such as adding protectants, to minimize the adverse effects of lyophilization for most proteins.
4. Why are protectants added to protein solutions before lyophilization? What protectants do you add to your products?
Protectants (stabilizers) are added to protect proteins during lyophilization and/or long-term storage. Proteins are subjected to various stresses generated by lyophilization which may cause loss of activity, aggregation, or denaturation. Protectants can alleviate the stresses by several mechanisms including the formation of an amorphous glassy state, the replacement of water, hydrogen bonding to proteins, and physical dilution and separation of protein molecules. Common protectants/stabilizers include sugars, polyols, polymers, surfactants, as well as some proteins and amino acids. We use trehalose and mannitol (normally 8% w/v) as protectants for lyophilization. Trehalose is a non-reducing disaccharide, well-known for its ability to stabilize biomolecules and microorganisms during prolonged period of desiccation. Mannitol is also commonly used as a stabilizer as well as a bulking agent in the process of lyophilization. It has been reported to reduce aggregation for some proteins during lyophilization.
5. How do I reconstitute the lyophilized powder? Should I use sterile water, PBS or some other buffer?
We recommend using sterile water for reconstitution. Add the recommended volume of sterile water to the vial and gently shake it to solubilize the protein completely. Make sure not to shake the vial too aggressively. PBS or some other buffer can also be used as the reconstitution agent as the salts in PBS can be ignored when the concentration and reconstitution volume is low. If possible, we suggest that you compare the results of different reconstitution agents.
6. Why are some proteins fused to tags?
The initial use of protein tags is for protein purification purpose. In addition, tags are also used for protein detection in several applications, such as western blot and ELISA, when the specific antibodies are not available. Furthermore, Fc tags stabilizes molecules, which may increase the half-life of the linked products. Since the Fc fragment tends to dimerize, it helps the linked protein, particularly receptors, to form biologically active dimers.
7. Do protein tags affect protein activity?
It is different from case to case. For some applications, small tags, such as His-tag, may not affect protein activity and do not need to be removed. For example, there are more than 100 structures of His-tagged proteins in the Protein Data Bank. This indicates that the small His-tag often does not interfere with correct protein folding. If there is no activity data online and activity is crucial to your experiments, please feel free to contact us (email@example.com) for latest information.
8. How should I store the products?
Lyophilized proteins are stable for at least twelve months from the date of receipt when stored at -20oC to –70oC. Upon reconstitution, the proteins can be stored at 2oC - 8oC for at least 1 month without detectable loss of activity. Reconstituted protein can also be aliquoted and stored frozen at -20oC to -70oC in a manual defrost freezer for six months without detectable loss of activity. Avoid repeated freeze-thaw cycles.
9. Can the products be used for other applications which are not listed on ACRObiosystems Inc. Certificate of Analysis?
If a specific application is not listed on the ACRObiosystems Certificate of Analysis and not tested by ACRObiosystems, please send us your requirements to firstname.lastname@example.org. We will check if it is possible to perform the assay or if any published data of the same product and the same application is available.
10. What should I know about the stability of your protein products?
Unless otherwise mentioned on the product information sheet, all of our products are formulated in such a manner that the lyophilized proteins are very stable at room temperature. However, we recommend storing lyophilized products at -20oC to -80oC.
For reconstituted solutions of most products, we recommend short-term storage at 4oC. For longer term storage the protein solution should be stored with a carrier protein (eg. 0.1% BSA) in working aliquots and stored frozen at -20oC to -80oC.
Please keep in mind that every freeze/thaw cycle may cause some denaturation of the protein.
11. What endotoxin level should be expected when purchasing ACRObiosystems proteins?
For most of ACRObiosystems proteins, the endotoxin level is guaranteed to be less than 0.1 ng/ug of protein, or 1 EU/ug. However, for many proteins, the actual measured endotoxin values are consistently below this stated endotoxin level.
Please contact Technical Support for more information.
12. Why can't I see the protein pellet in the vial?
Unlike many protein products available on the market, ACRObiosystems products are not formulated with carrier protein or other additives (e.g., BSA, HSA, sucrose, etc.) and are often lyophilized with a minimum amount of salt. As a result, the small amounts of protein can be deposited on the vial during lyophilization as a thin and sometimes invisible film. Before opening, we recommend centrifuging each vial in a microcentrifuge for 20-30 seconds to drive any protein that may be lodged in the cap or on the side to the bottom of the vial. Our quality control procedures assure that each vial contains the correct amount of product.
13. What is the relationship between the specific activity expressed as an ED50 and as units/mg?
The ED50 is defined as the cytokine concentration at which the activity is 50% of the maximum response. This method of expressing potency should only be used for cytokines whose dose-response curves are sigmoidal in shape. The formula for converting the activity as an ED50 in ng/ml to specific activity in units/mg is:
14. Who should I contact if I receive the wrong product quantity? How will you deal with this kind of complaint?
You can call us at 800-810-0816 or send us an email to email@example.com. We will repeat a quantification assay on the same batch of product that was sent to you. If there is a discrepancy between the two assays, we will perform more types of assays to determine the quantity. If the quantity is actually incorrect, we will make up the shortage for free and give you credits for your future orders.
15. How do I create a new online order?
To place a new order, search for your product using the Product Search Box on the top of the home page. Enter the quantity and click on the Add to Cart icon to begin the order process.
16. Can I get a discount?
We only offer discount to those who make bulk purchases. For bulk quotation, please contact us by email: firstname.lastname@example.org. Please indicate the approximate quantity of the product you want to purchase in your email.
17. How can I pay for my order?
For offline orders, the acceptable payment methods include PayPal, wire transfer and check. For online orders, all major credit cards are accepted in addition to the above methods. For details, please see ht
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