1. Q: Why is the shipping temperature for lyophilized products listed as room temperature?

A: Experimental data has demonstrated the excellent stability of DIMA’s lyophilized products at ambient temperatures. Domestic express shipping with a 2-3 day transit period does not adversely affect product activity.

2. Q: What is the concentration of trehalose in the lyophilized products, and does it impact their functionality?

A: Before lyophilization, our products are supplemented with a 5%~8% mass/volume solution of trehalose to ensure stability during the freeze-drying process. Post-lyophilization, we validate stability through methods like SDS-PAGE, and functionality through assays such as ELISA or FACS Binding. You can use our products with confidence.

1. Q: How should I reconstitute lyophilized products? How is the reconstitution volume calculated, and is it necessary to follow the pre-lyophilization concentration?

A: The Certificate of Analysis (COA) accompanying your order provides the pre-lyophilization product concentration and recommended dilution volume. Using a solvent concentration that is too low or too high can impact protein stability. Before use, it is essential to centrifuge the product and reconstitute it with sterile water. Our proteins are lyophilized from 1X PBS, pH 7.4, and typically contain 5% – 8% trehalose as protectants. Reconstituting based on the pre-lyophilization concentration yields the protein in 1X PBS buffer. Alternatively, using a larger volume of water may result in a lower salt concentration in the solution. While this is generally acceptable, we advise considering your specific experimental requirements when making this choice.

2. Q: Regarding the verification of product functionality, what analyses are available?

A: For some of our products, we currently provide verification data through ELISA or flow cytometry testing. Other activity analysis data may not be available at the moment. Please refer to the verification data charts on our website for the most up-to-date information.

3. Q: How is the stability of different batches of DIMA products?

A: For our popular proteins and antibody products, DimAb has implemented an efficient stable cell line development process internally, significantly minimizing batch-to-batch variations in product production. Our stringent quality management system ensures product quality, providing you with confidence in usage. If you encounter any issues during use, please feel free to contact us at any time.

1. Q: What is the endotoxin level in our product?

A: The production environment is controlled to minimize endotoxin introduction. Most of our proteins and antibodies are produced using HEK293 cells, a human cell expression system, rather than E. coli systems or other native resources. Most endotoxin concerns are linked to proteins prepared from E. coli. As a result, routine endotoxin testing is not part of our standard procedure. However, we can assure you that the endotoxin levels of our proteins are below 2 EU/μg (LAL).

1. Q: Can you send us the amino acid sequence of the proteins?

A: The amino acid sequence of our protein is available upon request. Please feel free to contact us, and we will promptly provide the requested information.

2. Q: Do you offer custom service for protein production?

A: Yes, we provide custom protein production services tailored to your specific needs. We’ll collaborate with you to design a project based on your sequence requirements. If you’re interested in a custom project, let us know, and we can provide details on pricing and timelines. Our dedicated team ensures the delivery of high-quality proteins meeting your research requirements. For any additional questions or assistance, please feel free to contact us.

1. Q: Can you let me know the extinction coefficient that was used for determining the concentration of the antibodies?

A: Due to the higher complexity of antibodies, we typically utilize the machine’s default extinction coefficient. The extinction coefficient used for determination of our full-length antibodies (HC+LC) is 0.735. The concentration was calculated using the formula A280/0.735. Generally, the predicted extinction coefficient aligns well with both UV and Bradford assay-determined concentrations, and we have verified this consistency.

2. Q: Can you provide more information about the antibody isotype mentioned on your website? Why do you have antibodies raised from Rabbit (Host Species: Rabbit) but with antibody Isotype: Rabbit/Human Fc chimeric IgG1?

A: This antibody is in a chimeric form. Initially developed with a rabbit, we then removed the rabbit Fc domain and replaced it with human IgG1 Fc. Hence, it is termed a Chimeric antibody, with the Fab portion being of rabbit origin and the Fc portion being human. This innovative design combines the advantages of rabbit antibody production with the compatibility of the human Fc region. For secondary antibody use, we recommend Goat anti-human-HRP or other conjugates.

3. Q: Will DIMA’s anti-DXD antibodies bind exatecan mesylate?

A: Our anti-DXD antibody will not bind exatecan mesylate. DIMA’s anti-DXD antibodies were originally developed by using DXD payload toxin. Exatecan Mesylate is closely related but not identical to DXd. They have structural differences.

1. Q: Which materials are used for Nanodisc Proteins?

        A: We utilize synthetic nanodiscs that are internally synthesized within our company. However, specific details regarding the composition of our nanodiscs are proprietary and confidential.

2. Q: Is it possible to purchase empty nanodiscs?

       A: Currently, we do not have empty nanodiscs available for purchase. However, we do offer nanodiscs designed for specific target proteins.

3. Q: Has the function of the protein/nanodisc been tested after lyophilization and dissolving?

       A: Yes, all our nanodiscs undergo comprehensive lyophilization and testing procedures. The data presented on our website is derived from the protein after the lyophilization and reconstitution process. The products are shipped at room temperature after lyophilization.

4. Q: Do you offer additional testing for standard Nanodisc products?

       A: Our Nanodisc proteins have been verified using an anti-Flag tag antibody. We can attempt verification using other antibodies if suitable ones are available. However, please note that for some products, we may not have an appropriate antibody in our inventory. If you have any suggestions, kindly share them with us so that we can explore the possibility of working with the recommended antibody.

5. Q: Do you offer custom Nanodisc Development?

       A: We offer custom Nanodisc development for targets beyond our current range. To provide accurate costings and timelines, we require specific details about the target and the scale of the project. Please share your specific requirements to enable us to offer a precise estimate and timeline.

For new targets, we typically conduct a test run (80mL culture volume) with the expression cDNA clone. The obtained protein from the test run, varying from ng to 100ug, will be sent to customers. The actual cost and timeline depend on specific project requirements. For instance, a protein with 12 transmembrane domains, like MDR1 (Human MDR-1 full-length protein-synthetic nanodisc – DIMA Biotechnology), will incur a higher cost for the test run compared to a protein with 4 transmembrane domains, like CLDN2 (Human CLDN2 full-length protein-synthetic nanodisc – DIMA Biotechnology).

Additionally, any other considerations for a custom project, such as unique specifications or special handling instructions, can be discussed based on the project details. The expression cDNA clone will be charged separately if not provided by customers, and we will send the DNA clone to customers in this case.

6. Q: There are multiple bands in the Nanodisc sample.

      A: We are actively optimizing our conditions to achieve better results for this protein. However, based on our experiences, proteins can exhibit bands at higher molecular weights due to post-translational modifications (PTMs) such as phosphorylation and glycosylation, or bands at lower molecular weights due to cleavage during cellular processes or sample preparation. Therefore, we suggest checking the literature to see if multiple bands are reported for the specific protein. If you come across any reference data for this protein, please feel free to share it with us so that we can further optimize our conditions.

7. Q: How much protein can you typically obtain from an 80 ml culture for VLP, MNP, or Nanodisc preparation?

     A: The yield varies by method. For VLP, you can typically expect around 1 mg, although this can depend on the specific target, as some may express poorly. MNP usually yields about 2-3 mg. Nanodisc yields are lower, generally in the microgram range, which makes it relatively high-cost. However, due to its small size, Nanodisc can be purified via anti-tag antibodies, which results in high purity and homogeneity of final protein sample. In contrast, VLP and MNP are larger, meaning that while recombinant proteins can be enriched, they also contain some adjacent membrane proteins and molecules, resulting in lower purity compared to Nanodisc proteins.

8. Q: What is the total amount of protein and corresponding lipid and polymer? What is the protein measurement method you used for quantification?

     A: For Nanodisc protein measurement, we use standard BCA method. The amount indicated on the tube is the target protein amount excluding lipid and synthetic polymers. The molecular weight of lipid and polymer on synthetic Nanodisc is in average around 146kDa. Therefore, the amount of these two component can be calculated with following equation: The amount of lipid+synthetic polymer=amount of protein X (146/molecular weight of target protein). 

9. Q: What is the molecular weight of empty synthetic Nanodisc copolymer complex?

     A: The average molecular weight is around 146kDa. The detailed calculation can be referenced at this paper (Hall SCL., et al. 2018).

10. Q: Have any of your customers used synthetic nanodisc membrane proteins in display experiments? Which immobilization methods would you recommend? According to Dima Biotech webside, nanodisc membrane proteins have been tested in SPR and ELISA applications – how were they immobilized on solid phase in these applications?

        A: In terms of immobilization, we normaly use standard ELISA coating approach. It works fine. We also tried with sanwich approach, which first coat with anti-FLAG antibody, then Nanodisc protein. Both approaches work well. However, there are a couple of cases that certain Nanodisc protein might not work with direct coating approach when tested with conformational epitope antibody. For linear epitope, like anti-FLAG, it works all the time with direct coating approach or indirect sanwich approach.

11. Q: Do you offer biotinylated nanodisc membrane proteins?

        A: Currently we do not offer as a standard product format. However, we have developed the technology platform and can offer as a custom service project for you.

12. Q: Could you please provide more information on the stability, storage conditions and buffer compatibility of your polymer-based nanodiscs? For example, are they compatible with buffers containing detergents, e.g. Tween-20, or low concentrations of organic solvents e.g. DMSO or ethanol? Are there any other limitations when developing the assays with polymer-based nanodisc membrane proteins?

       A: Our copolymer is SMA based technology. Detergent could potentially affect the Nanodisc. However, we used the low concentration Tween-20 in the buffer during ELISA assay. So far, it is OK. In terms of reaction buffer pH, we recommend it should not be lower than pH5.0. SMA copolymer is sensitive to low pH condition. High concentration of divalent cations will also affect SMA based copolymer. We suggest you do not include more than 5mM concentration of divalent cations. We did not test experimental condition in DMSO or ethanol.

13. Q: Purity and purification methods of nanodisc

        A:  We used M2 affinity purification approach to purify the protein.According to the SDS-PAGE gel, the purity of the protein is estimated to be around 85%, even though there are faint high MW bands above the major product band, which is normal feature for transmembrane protein SDS-PAGE gel. The origin of these high MW bands could be due to multimers or PTM caused. If needed, we can run a WB assay to verify the authenticity of these high MW bands.

14. Q: What issues should be paid attention to when using your Nanodisc protein in ELISA and WB experiments?

        A: Due to the slight shedding of light/heavy chains of anti-Flag M2 antibodies during purification, we are unable to use anti-mouse secondary antibody for Flag-tag nanodisc in indirect ELISA or WB experiments. 

1. Q: When the test antibody is diluted and mixed with AME100002 according to the mass ratio, the Ab-AME100002 complex is formed. Can the labeled antibody be stored, and what are the storage conditions?

A: We have not conducted specific tests on this. We recommend preparing and using the labeled antibody immediately.

2. Q: What is the principle behind DiTag^TMpH-sensitive IgG labeling reagent, and how is the internalization rate calculated?

A: DiTag^TM pH-sensitive IgG labeling reagent utilizes a pH-sensitive fluorophore-labeled Fc-binding protein that binds to IgG antibodies from different species, forming a fluorescently labeled antibody-reagent complex. Upon internalization of the antibody, the surrounding pH becomes acidic, significantly enhancing the fluorescence signal of the antibody-reagent complex. The fluorescence intensity serves as an indicator of antibody internalization activity. By measuring the strength of the fluorescence signal, researchers can assess the efficiency of antibody internalization into cells. The positivity rate is calculated based on the customer’s experimental design, representing the percentage of positively internalized cells.

3. Q: For DiTag^TMpH-sensitive IgG labeling reagent, what is the achievable Mean Fluorescence Intensity (MFI)? How much difference in cell signaling values is considered significant? Are there cells that may not be detectable?

A: The achievable Mean Fluorescence Intensity (MFI) for DiTag^TM pH-sensitive IgG labeling reagent can vary based on different cell types and flow cytometry setups. There is a risk of not detecting antigen-low expressing cells. The MFI values can differ for various cell types, and the experimental data from DIMA Biotechnology indicates an MFI of FITC at 3*10^4.

4. Q: What is the difference between AME100001 and AME100002?

A: The main difference lies in their affinity for different IgG antibodies. Please refer to the table below for more information.