Recombinant Bifunctional ligase/repressor BirA
Why do proteins need to be lyophilized? What are the effects on proteins?
Proteins are heat sensitive and lyophilization allows the protein to be stored with maximum activity, extended shelf life, and reduced transportation costs. The effects of lyophilization on proteins include the possibility of partial inactivation, aggregation and other problems. However, these negative effects can be minimized by adding protective agents, stabilizers, additives, antioxidants and controlling the various conditions of lyophilization.
Why add protectants to the protein solution before lyophilization? What are the protective agents commonly used by PrimeGene?
Freeze-drying will have a certain effect on the activity of proteins, and the addition of a protective agent can reduce this effect and maximize the protection of protein activity. The usual protectants or stabilizers are sugars, polyols, certain proteins and amino acids. Our commonly used protective agents are: trehalose, Tween, etc.
The lyophilized form of the recombinant protein shipping at ambient temperature and the instructions indicate that it should be stored at -20ºC. Does this shipping method affect the activity of the product?
The lyophilized powder product will not be affected when shipped at ambient temperature. Freeze-drying improves the stability of the protein product while reducing packaging materials and shipping costs. We have tested the shipping conditions of our freeze-dried products to ensure product quality.
What is Trehalose and what is the function of Trehalose in the formulation?
Trehalose is a non-reducing sugar that does not react with amino acids or proteins in the Maillard reaction. It is found in many plants and animals in nature. Trehalose stabilizes proteins against damage caused by freezing and also makes them more water-tolerant when freeze-dried, thus making it less likely that the product will precipitate upon re-solution.
Does trehalose interfere the conjugation reaction?
The presence of trehalose may interfere with the successful binding of proteins. This will depend on the method of conjugation and customers are advised to do the relevant research before purchasing the product.
What does "mg/g" mean on the product label?
The quality of recombinant protein is not necessarily the same as that of freeze-dried powder because of the addition of protective agents or stabilizers to the solution before freeze-drying. Mg/g refers to the mass (mg) of recombinant proteins per gram (g) of freeze-dried powder.
Can the protein be used for in vivo experiments? What is the recommended concentration?
PrimeGene Protein does not perform any in vivo testing. You are advised to look for proteins that have been tested for biological activity, and determine the working concentration by reviewing the references for the in vivo application of the product.
If the product contains trehalose, will in vivo experiments affect the animals?
Trehalose is unlikely to affect in vivo experiments. It has been approved for use as an adjuvant in human injectable drugs. The trehalose used in PrimeGene is derived from Saccharomyces cerevisiae and has a purity of at least 98.5% as determined by HPAE.
In what application can the product be used?
Our products can be classified according to scientific fields, but no clear guidance is given on their specific applications. Since proteins can be explored for multiple applications and methods of application according to their functions, we recommend our customers to conduct experiments according to the literature or to explore their own application methods based on scientific research.
Why do some proteins come with labels?
The tagged proteins are generally used to meet the experimental requirements of some customers. For example, affinity columns can be used for His-tag and GST-tag; FC fusion proteins can be used for antibody studies, etc.
The amino acid sequence of some products like 1002-08 are not shown on the website, , Can they be provided by PrimeGene?
If the amino acid sequences of some products are not shown on the website, they may not be provided as confidential information, but some products can provide protein Accession# and GeneID for reference. If you have any questions, we recommend you to call the customer service hotline at 400-821-3475.
How is the protein quantified?
Usually three methods, BCA , Bradford and OD280, are used for quantification.
Why the results measured by my quantification method do not match the quantification on the PrimeGene label?
Different protein quantification methods will give the different results. Sometimes the difference in results caused by different quantification methods is significant. Each detection method has its limitations, so appropriate detection methods should be chosen for different proteins. In addition, the sample dilution process will also affect the results. For example, dilution directly from high concentration to low concentration (mg-ug-ng) may have greater errors than 10 times gradient dilution; and the use of consumables such as non-low adsorption item, may also cause sample concentration deviation; we do not recommend the quantification for trace proteins due to uncertain factors introduced in the dilution process.
I purchased a small package of lyophilized protein, why I received a empty tube?
Unlike some protein products on the market, most of our products do not contain carrier proteins or other additives (such as bovine serum albumin (BSA), human serum albumin (HSA) and trehalose) and are lyophilized in a solution with minimal salt content. As a result, trace amounts of protein are deposited inside the tube during the lyophilization process, forming a very thin or almost invisible protein layer, and even the protein powder can drift on the tube walls. Before opening the cap, we recommend rapid centrifugation in a centrifuge for 20-30 seconds to allow the protein powder attached to the cap or wall to collect at the bottom of the tube.
How to reconstitute the lyophilized powder protein?
Unless the instructions are more specific, we recommend the following.
(1) Equilibrate the reagent bottle and reconstitute buffer to room temperature.
(2) Centrifuge the reagent vial briefly to ensure that all lyophilized material is collected at the bottom of the vial.
(3) Add the recommended re-dissolution buffer to the concentration recommended on the product description. Avoid vigorous shaking or pipette blowing, which may result in denaturation of the protein.
(4) Allow reagent vials to redissolve at room temperature for 15-30 minutes, either using a shaker or manually inverting up and down gently.
(5) Dispense in volumes no less than 10μL per tube and store in accordance with the instructions in the product manual.
(6) If the vial presents flakes or pellets, mix the product at room temperature for several hours and then overnight at 4°C.
What is the reconstitution concentration of PrimeGene protein products?
The "Reconstitution" section of the product page can be used for your reference. For example, product cat#105-02 is in the form of freeze-dried powder, and reconstitute in sterile distilled water or aqueous buffer containing 0.1 % BSA to a concentration of 0.1-1.0 mg/mL. In addition, we usually recommend high quality BSA (high purity, low endotoxin, low fatty acid and can be used for cell culture). For short-term use, it can be directly dissolved in sterile distilled water, if sub-package cryopreservation is needed, we recommend to add 0.1%BSA to avoid protein degradation. Besides, PrimeGene also provide some high concentration products, such as cat#105-02G, which is in the form of freeze-dried powder. The recommended reconstitution concentration is 0.1-5.0mg/mL in sterile distilled water or aseptic aqueous solution containing 0.1%BSA.
How to determine the reference dosage for protein activity experiments?
All proteins which have been verified by biological activity have activity verification information on our product page, customers can check the information of cell type and ED50 value in the Biological Activity column. The methods of activity detection are different for different proteins. The methods used to detect different cells for the same protein are different. The final ED50 value is also different. In the absence of literature and reference dosage, we recommend trials for the concentration gradient in your initial use of a certain protein.
Why is ED50 a range?
Biological activity is a measurement of the biological response of a protein. Therefore, any changes in the bioactivity assay may also lead to different results. Such variations may include cell density, cell age, number of passages, nutritional status, media and additives used, as well as the experimental operator’s practices. Due to the variable nature of bioactivity assays, the same protein may not produce the same ED50 under different circumstances. For this reason, PrimeGene’s protein instructions give a range of ED50s.
How is your protein activity measured?
PrimeGene’s activity-verified protein products are usually listed as Biological Activity in the technical parameters of the product page. For example, product cat#GMP-606-09 shows activity measured by its ability to support cell attachment and spreading when used as a substratum for cell culture. In this application, the recommended concentration for this effect is typically 1-5 μg/cm². Fibronectin can also be added to the media to support cell spreading at a concentration of 0.5-50 μg/mL.
What is the relationship between the specific activity marked by ED50 or units/mg, and how to convert it?
The definition of ED50 is the cytokines concentration that can induce the half of the maximum response. This activity expression is only applicable to cytokines with an S-shaped dose-response curve. The formula for transforming ED50 (ng/ml) into specific activity (units/mg) is:
During the reconstitution of dry protein powder, it is mentioned in the manual that acid is used to dissolve, will it affect the activity of protein? When used in cell experiments, will it cause damage to the cells?
The dissolution method recommended in the instructions for the use of protein will get the best dissolution effect and ensure the most complete recovery of protein activity. A small amount of acid will not affect your experiment, after the original solution is diluted into a working solution when it is used.
How to store the product?
Because of the different nature of the products, please read the instructions carefully and store them properly according to the methods described in the instructions.
What is the difference between GMP protein and traditional protein?
In general, the sequence and expression system of GMP protein is the same as that of traditional protein. The GMP protein of PrimeGene ® is made from the relevant section of the ICH-Q7 a Good Manufacturing Practice Guide for Active Pharmaceutical ingredients file and has the following advantages: 1. Detailed product-related information; 2. Strict quality management system; 3. High stability between batches; 4. No exogenous animal origin; 5. Can provide relevant laws and regulations support documents as needed according to our customer needs; 6. Production equipment and production environment comply with relevant laws and regulations and GMP guidelines; 7. Products for aseptic testing, no mycoplasma, HCP detection; 8. The process and detection methods of production equipment have been verified; 9. Raw material testing and supplier qualification review; 10. Perfect material review process and documentation management, production personnel qualification in line with the requirements of laws and regulations