Protein FAQs

Important Questions

Q: What products or services do you offer?

CUSABIO provides 70 native proteins, 100 small molecule antigens, 320+ active proteins, 1000+ recombinant proteins in stock, 5700+ developed recombinant proteins, 36,000+ transmembrane proteins, 500,000+ semi-customized recombinant proteins, and also could provide risk-free protein expression service.

Q: Why do we choose your proteins?

CUSABIO provides "No risk" custom service for millions of highly purified recombinant proteins in different species with different scales by different expression systems.

We have established five recombinant expression systems from prokaryotic (E. coli) to eukaryotic (Yeast, mammalian cell and insect baculovirus), and has also built unique In vitro E. coli expression system, which enables us to express transmembrane proteins that are usually quite difficult to express. Furthermore, CUSABIO protein QC department owns a professional technical team, equipped with advanced experimental apparatus, to ensure each protein has a complete COA report and high quality. CUSABIO’s purified proteins surpass 85% purity as detected by SDS-PAGE analysis.

Q: Where can I find references from other researchers that have used your proteins?

Our official website has updated the articles that researchers who have published their results after using CUSABIO proteins. Please click here to see more:

Application and Selection of Our Products

Q: What are the applications of recombinant proteins?

Recombinant proteins have wide applications in medicine, basic research, and biotechnology. You can learn more from this link:

Q: Which expression system should we choose in priority order?

We have five expression systems including E. coli expression system, In vitro E. coli expression system, Yeast expression system, Insect baculovirus expression system and Mammalian expression system. They are different in the post-translation modification and the choice depends on your purpose of experiment. E. coli expression system is widely studied and cheap to purchase, which is right choice for immune-related experiments, receptor and ligand binding experiments, cell experiments or drug screening experiments, enzyme activity experiments refer to each relative species. Click here to learn more about CUSABIO's Five Expression Systems.

Q: What validations have you performed for this protein?

Normally, we provide molecular weight, electrophoretic results (SDS-PAGE), concentration, purity, etc. If you have special demand, we can also provide electrophoretic parameters, activity evaluation, sequence identification, tag removal service, endotoxin removal service, WB and MS validations. You can learn more from this link:

Q: What is the concentration range of your proteins? How do you determine the quantity of them?

The protein concentration of each batch won't be exactly the same but we could guarantee 0.1-5.0 mg/mL concentration for our expression system. If you have special requirement for protein concentration, please communicate with us in advance.

There are three methods of protein concentration detection: bradford method, BCA method, A280 method, and each of these three methods has interference buffer components. According to the composition of buffer, the bradford method is the most popular with laboratories including ours, and the concentration detection range is 0.1-5 mg/mL. Sometimes, we also use BCA method.

Q: What is the purity of each protein?

The purity is higher than 85% as determined by SDS-PAGE.

Q: Have you done activity test of each protein?

All of our proteins are purified under mild conditions. If a protein we provide is full length of mature protein, then it should have activity in theory. Since the activity is not 100% guaranteed, we suggest the customer to purchase small size to do validation first. If the protein is active after their validation, they could purchase larger size later, and we will provide certain discount for their re-order. We also have some proteins that have been verified for their activity and we have listed their biological activity data on our website. Click here to learn more about corresponding protein for details.

Q: What specifications do you have in your products?

Proteins with different sizes (5 μg, 10 μg, 20 μg, 50 μg, 100 μg, 500 μg, 1 mg, etc) are available. Additionally, we have good capacity and experience for large-scale expression (10 mg, 50 mg, 100 mg, 200 mg, 500 mg, etc).

Q: What types of tags do you use for fusion?

The common tags we provide include His-tag, FLAG-tag, GST-tag, MBP-tag, combination tags (His-GST-tag, His-sumo-tag, His-MBP-tag), etc. Sometimes, the tag of proteins will be determined during the manufacturing process. If you have specified tag type, please feel free to consult with us. Click here to learn more about the general information of different tags.

Q: What are the sequences of the tags?

The sequence varies from tag to tag. Please contact us for details.

Q: What is the molecular weight of the recombinant protein?

The theoretical MW of the fusion protein = The MW of the tag + The MW of the target protein. You can use CUSABIO Molecular Weight Calculator to calculate the theoretical value or refer to the Datasheet & COA upon shipment.

Q: What is the impact of a given tag type and any potential biological activity of the protein?

Theoretically small tags generally have very small influence on protein activity. However, the specific impact on protein activity can't be concluded (There is no impact on some proteins, small impact on some proteins, and relatively great impact on some proteins).

Q: Can you remove the tag?

Not all protein tags can be removed as some proteins will be very unstable after tag removal.

Please communicate with us in advance if you need to remove the tag which takes 2-3 business days. If we succeed in removing the tag, we will charge for extra cost for tag removal. If we fail in removing the tag, we won't charge for any extra cost, and remark this information in datasheet as follows "Note: The laboratory determined that the tag on your protein could not be removed with standard laboratory procedures. Your protein is being supplied with the tag intact."

Q: Can you remove the endotoxin?

Not all endotoxin can be removed. Please communicate with us in advance if you need to remove the tag which takes 2-3 business days. We could offer endotoxin removal service free of charge using PMB affinity chromatography, use LAL reagent to semi-quantitatively detect the content of endotoxin and guarantee endotoxin level within 0.1 ng/μg (1 EU/μg).

Q: Can you offer aseptic manufacture processing?

Yes, we can offer this service and it is free of charge, but you should remark this information when placing the order. We've performed aseptic processing for liquid protein before lyophilization, but there may exist contamination during lyophilization process, so we can't say germ-free for the whole process.

Q: Can you provide corresponding antibody for this recombinant protein?

Yes. We could provide corresponding antibody for the recombinant protein. Based on the quotation for the recombinant protein, add certain cost, we will offer 10-15 mg affinity-purified polyclonal antibody with a guarantee of western blot and ELISA positive on this recombinant protein. The delivery time will be 2.5 months after the delivery of the protein. Please feel free to contact us to get more details.

Q: Why are our protein products almost invisible in pipes?

CUSABIO protein product does not contain carrier protein or other additives (such as bovine serum albumin (BSA), human serum albumin (HSA) and sucrose, and lyophilized from low salt solution, so it often does not form a white grid structure, but a trace amount of protein deposit within the tube, forming a thin transparent or invisible protein layer.

Tips: Before opening the lid, we recommend to centrifuge in a small centrifuge for 20-30 seconds firstly to ensure that the contents are on the bottom of the tube. Our quality control steps ensure that the amount of protein contained in each tube is accurate, although sometimes you can’t see the protein powder, but the protein content in the tube is still very accurate.

Q: How to determine species cross-reactivity of cytokines?

a. Apart from a few exceptions, most human cytokines are active on mouse cells.

b. Many mouse cytokines may also have effect on human cells, however, the activity may be lower than the corresponding human cytokines.

c. One of the few human cytokines will be more active than corresponding mouse cytokines when acting on mouse cells, such as IL-7.

d. Interferon, GM-CSF, IL-3 and IL-4 and other cytokines are species-specific and almost have no activity on non-homologous cells.

e. In contrast, fibroblast growth factor (FGF) and neurotrophin are highly conserved and both have good activity on cells of different species.

Q: What is the general preservative? Which kind of preservative do you usually add?

Commonly used preservative include Proclin 300, Sodium azide, etc. We do not add any preservative to our proteins.

Q: What is the general protectant? What kind of protectant do you usually add?

Commonly used protectant include saccharides, polyols, polymers, surfactants, some proteins and amino acids etc. We usually add 8% (mass ratio by volume) of trehalose and mannitol as lyoprotectant. Trehalose can significantly prevent the alter of the protein secondary structure, the extension and aggregation of proteins during freeze-drying process; mannitol is also a universal applied protectant and fillers, which can reduce the aggregation of certain proteins after lyophilization.

General Troubleshooting Guide for Protein Synthesis

Q: What is protein expression and purification?

Protein expression is the biotechnological process of generating a specific protein. It can be done in prokaryotic, eukaryotic or In vitro E. coli expression system. Protein purification is a series of processes intended to isolate one or a few proteins from cells or organisms. The most popular method for protein purification is affinity chromatography, and which is designed by different protein tags. Other protein purification methods, including ion exchange chromatography, size-exclusion chromatography, polish purification and hydrophobic interaction chromatography are available to handle tag-free proteins with high purity.

Q: Why is there no/low protein expression?

a. Incorrect vector construction. You should confirm vector by sequencing or apply for our custom clone service.

b. Rare codons. You should optimize codons, use strains supplementing rare codons, induce at lower temperature or grow in poor media.

c. Protein toxicity. You should use promoters with tighter regulation or lower plasmid copy number. Use pLysS/pLysE bearing strains in T7-based systems or strains that are better for the expression of toxic proteins. Start induction at high OD and shorten induction time. Add glucose when using expression vectors containing lac-based promoters.

Q: How to avoid inclusion bodies and improve soluble expression?

a. Proteins with high hydrophobicity or transmembrane domains. You should add fusion tags or add heat shock chaperones. You should induce for a shorter time at low temperature or change to poor media. Generate truncated forms of protein or use membrane rich strains.

b. Incorrect disulfide bond formation. You should add fusion partners, including thioredoxin, DsbA, DsbC. Clone in a vector containing secretion signal peptide to cell periplasm. Use gamiB (DE3)strains with oxidative cytoplasmic environment. Lower inducer concentration and induction temperature.

c. Incorrect folding. You should use a fusion partner. Co-express with molecular chaperones. Use strains with cold-adapted chaperones. Supplement media with chemical chaperones and cofactors. Reduce the inducer concentration and add fresh media. Induce for a shorter time at low temperature.

Q: Why is the molecular weight of protein smaller than the predicted?

a. Rare amino acids selenocysteine (Sec) or pyrrolysine (Pyl) in protein sequence. You should use some other amino acids to instead these two unusual amino acids.

b. Imbalanced translation process of fusion protein. You should change another fusion tag or move fusion tag to C-terminal. You should induce for a shorter time at low temperature or change to poor media.

c. Protein degradation. You should replace specific protease sites. Use protease deficient strains. Induce at high OD. You should induce for a shorter time at low temperature or use protease inhibitors when breaking cells.

Q: Why is the actual band size different from the predicted?

a. Post-translational modification. Phosphorylation, glycosylation, etc which increases the size of the protein.

b. Post-translational cleavage. Many proteins are synthesized as pro-proteins, and then cleaved to give the active form.

c. Splice variants. Alternative splicing may create different sized proteins from the same gene.

d. Relative charge. The composition of amino acids have different relative charge which will affect the electrophoretic mobility.

e. Multimers such as dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands.

f. Protein structure such as disulfide bond, protein secondary structure or protein 3D structure formation.

g. Hydrophobic proteins, such as transmembrane proteins, may have difficulties in migrating into the gel, and thus resulting in different multi-banded patterns.

Q: How to express a protein with bioactivity? Why is the protein inactive?

For gaining a protein with bioactivity, you should choose a right expression system, a suitable expression vector, an appropriate purification method and a validation experiment. You can learn more from this link: Otherwise, you can check the problems below:

a. Low solubility of the protein. You should fuse desired protein to a fusion partners and lower temperature.

b. Lack of essential post translational modification. You should change another expression system.

c. Incomplete folding. You should use a fusion partner and use strains with cold-adapted chaperones. Co-express with molecular chaperones at lower temperature. Monitor disulfide bond formation and allow further folding in vitro.

d. Mutations in cDNA. You should sequence plasmid before and after induction or use a recA strain to ensure plasmid stability. Transform E. coli before each expression round.

Shipping and Storage

Q: How long can I get the products after I place an order?

Most of our protein products are in stock and can be shipped out in 3-7 business days once passing our Quality Control process. Each of our proteins will undergo strict QC process before be delivered.

Custom protein service takes about 1 month, Click here to learn more about five recombinant expression systems.

Q: What could be the deliverables for your protein expression service?

a. Purified protein, the purity is more than 85%.

b. Standard COA report as well as datasheet, including tag information, molecular weight, electrophoretic parameters, protein expression quantity, concentration, purity, SDS-PAGE, etc.

Q: What is the delivery form?

The default delivery format of protein is liquid form and the default storage buffer is Tris-based buffer, pH=8.0, 50% glycerol. If you have special requirement for the storage buffer, please feel free to contact us.

Though the lyophilized form will be more stable than the liquid form, some proteins structure and activity may be damaged by the lyophilization process. We add protective agents and control lyophilized condition to reduce the damage. If the customer has demand for lyophilized form, please remark this requirement when placing order.

Q: How should I reconstitute and store the products?

Centrifugate the reagent tube before opening the cap.

As for short-term storage or usage, please use sterile deionized water to completely reconstitute proteins to 0.1-1.0 mg/mL. Aliquot after 10-15 minutes if needed and store at 4℃.

As for long-term storage, the cytokines or recombinant proteins are recommended to add 5-50% of glycerol (final concentration) and aliquot for long-term storage at -20℃/-80℃. Our default final concentration of glycerol is 50%. Customers could use it as reference.

Q: What is the shelf life of the products?

The shelf life is affected by many factors such as storage state, storage temperature, buffer ingredients and the stability of the protein itself.

Generally, the shelf life of liquid-form protein is 6 months around at -20℃/-80℃, and the shelf life of lyophilized form is 12 months at -20℃/-80℃.

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301-363-4651 (Available 9 a.m. to 5 p.m. CST from Monday to Friday)
7505 Fannin St. Ste 610-312, Houston, TX 77054, USA
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