Recombinant DT3C (Diphtheria toxin & spg 3C domain) for Antibody Internalization Assay (Active)

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Code CSB-EP360556CQR1
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  • (Tris-Glycine gel) Discontinuous SDS-PAGE (reduced) with 5% enrichment gel and 15% separation gel.
  • Activity
    After DT3C 10μg/ml formed complexes with different concentrations of Anti-CCR8 antibodies (CSB-RA004847MA3HU), cytotoxicity experiments were conducted on CHOK1/Human CCR8 cells (CSB-SC004847HU2). The ED50 is 0.3708-0.5608 μg/ml. Biological Activity Assay
  • Activity
    After DT3C 10μg/ml formed complexes with different concentrations of Anti-CCR8 antibodies (CSB-RA004847MA3HU), cytotoxicity experiments were conducted on CT26/Human CCR8 cells (CSB-SC004847HU3). The ED50 is 0.4661-0.8614 μg/ml. Biological Activity Assay
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Product Details

Purity
Greater than 90% as determined by SDS-PAGE.Greater than 90% as determined by SEC-HPLC.
Endotoxin
Less than 1.0 EU/ug as determined by LAL method.
Activity
①After DT3C 10μg/ml formed complexes with different concentrations of Anti-CCR8 antibodies (CSB-RA004847MA3HU), cytotoxicity experiments were conducted on CHOK1/Human CCR8 cells (CSB-SC004847HU2). The ED50 is 0.3708-0.5608 μg/ml.②After DT3C 10μg/ml formed complexes with different concentrations of Anti-CCR8 antibodies (CSB-RA004847MA3HU), cytotoxicity experiments were conducted on CT26/Human CCR8 cells (CSB-SC004847HU3). The ED50 is 0.4661-0.8614 μg/ml.
Target Names
DT3C
Uniprot No.
Species
N/A
Source
E.coli
Expression Region
33-417aa(P00588)+linker(GGGGSGGGGSGGGGS)+291-497aa (P19909)
Target Protein Sequence
GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKGGGGSGGGGSGGGGSIDEILAALPKTDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE
Mol. Weight
69.4 kDa
Protein Length
Linked Heterodimer
Tag Info
N-terminal 6xHis-tagged
Form
Lyophilized powder
Note: We will preferentially ship the format that we have in stock, however, if you have any special requirement for the format, please remark your requirement when placing the order, we will prepare according to your demand.
Buffer
Lyophilized from a 0.2 μm filtered PBS, 6% Trehalose, pH 7.4
Reconstitution
We recommend that this vial be briefly centrifuged prior to opening to bring the contents to the bottom. Please reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.We recommend 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.
Troubleshooting and FAQs
Storage Condition
Store at -20°C/-80°C upon receipt, aliquoting is necessary for mutiple use. Avoid repeated freeze-thaw cycles.
Shelf Life
The shelf life is related to many factors, storage state, buffer ingredients, storage temperature and the stability of the protein itself.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Lead Time
Basically, we can dispatch the products out in 3-7 working days after receiving your orders. Delivery time may differ from different purchasing way or location, please kindly consult your local distributors for specific delivery time.
Notes
Repeated freezing and thawing is not recommended. Store working aliquots at 4℃ for up to one week.
Datasheet & COA
Please contact us to get it.
Description

The recombinant DT3C protein was produced by expressing a DNA fragment encoding the catalytic and translocation domains of the Corynephage beta diphtheria toxin (DT) (33-417aa) fused with the Streptococcal protein G-derived 3C domain (291-497aa) in E.coli. The 6xHis-tag sequence was placed in the N-terminus of the DT3C encoding sequence. The resulting recombinant DT3C protein consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C) [1]. The purity of the recombinant DT3C protein was assessed using SDS-PAGE, demonstrating a purity level of up to 90%. On the gel, DT3C migrated as a band with an approximate molecular weight of 69.4 kDa.

The rationale behind designing the recombinant DT3C protein stems from the cytotoxic property of diphtheria toxin, which inhibits the protein translation machinery in cells, and the Fc-binding domain (3C), capable of binding to any IgG antibodies across different species [1][2]. It binds to a mAb to form a mAb-DT3C conjugate after incubation at room temperature, which functions in vitro similarly to an antibody-drug conjugate (ADC), delivering the cytotoxic effects of diphtheria toxin specifically to targeted cells. Moreover, the mAb-DT3C conjugate serves as a tool to assess the efficiency of mAb internalization by cells by monitoring the extent to which it induces cell death. Additionally, the recombinant DT3C protein facilitates the evaluation of cancer cells' internalization efficiency of the mAb in vitro, aiding in the screening process for more effective monoclonal antibodies.

References:
[1] Hamamichi, S., Fukuhara, T., et al. Novel method for screening functional antibody with comprehensive analysis of its immunoliposome [J]. Sci Rep 11, 4625 (2021).
[2] Miki Yamaguchi, Yukari Nishii, et al. Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells [J]. Biochemical and Biophysical Research Communications, Volume 454, Issue 4, 28 November 2014, Pages 600-603.

Customer Reviews and Q&A

 Customer Reviews
Average Rating:
5.0 - 4 reviews

Submit a Review here

Applications : Cell assay

Review: I purchased DTC3 recombinant protein for hybridomas screening and referred to the experimental process in a certain literature, and the results were very satisfactory. The specific experimental process is as follows: 4 μg/mL DT3C (25 μL) was added to a 96-well plate, and the culture supernatant of the hybridomas obtained (25 μL) was further added, and the resultant was incubated at room temperature for 30 minutes. NOR-P1 cells (50 μL) were seeded at 2 x 10^5 cells/mL (RPMI medium 10% Low IgG FBS (+)), and cultured in a CO2 incubator at 37℃ for 3 days. Through microscopic observation after culturing, wells with the number of adhering cells being about 25% or less of that in using a negative control antibody were determined to be positive.Selected clones were subjected to one or two subcloning steps to establish eight monoclonal hybridoma cell lines.

By Anonymous

Applications : /

Review: I purchased CSB-EP360556CQR1 for antibody internalization assay and followed the detailed experimental procedures recommended by CUSABIO. The experimental process was very detailed and the service was also very attentive. I tested DT3C in different cells and showed different effects. I found that different types of cells have different levels of sensitivity to DT3C, with some showing better results in the antibody internalization assay. The efficiency of antibody internalization is closely related to the antigen density, antibody affinity, and antibody and antigen-binding epitopes. Different antibodies to the same target will also show different internalization efficiencies. Therefore, it is recommended to test several more cells in the early stage.

By Anonymous

Applications : /

Review: We purhcased CUSABIO\'s DT3C product for detecting antibody internalization, compared with other methods, its mAb-DT3C complex preparation is simple, and its antibody internalization efficiency is higher than Mab-ZAP, with high cost-effectiveness. We will purchase again.

By Anonymous

Applications : Cell assay

Review: I did the endocytosis experiment with CSB-EP360556CQR1, and the effect was very good.

By Anonymous

 Q&A
Q:

I am looking at the DT3C internalization email. I have a drug that works like an ADC, but everything in front of the linker is synthetic, will this assay work for measuring internalization and also internalization rate?

A:

DT3C mainly measures the internalization efficiency of antibodies, it doesn’t work for measuring internalization rate of ADC analog.
CUSBAIO recombinant DT3C (Diphtheria toxin & spg 3C domain includes a diphtheria toxin (DT) without a receptor binding domain and the C1, C2, C3 (3C) domains of Streptococcus protein G, that is DT3C. DT3C is an effective tool to detect the efficiency of mAbs' internalization by cells after Mab-ZAP. There is study has revealed that, compared with mAb-Mab-ZAP, mAb-DT3C mimics antibody-drug conjugate (ADC) with these features, including easy preparation, molecular weight stability and high efficiency of antibody internalization. According to incomplete statistics, DT3C has been used in nearly 130 ADC drug mAb screening patents as a tool to detect cells' ability of mAb internalization.

Q:

We have ordered 1mg of your recombinant DT3C protein (CSB-EP360556CQR1) and are very satisfied with the testing result, we would like to place a bulk order of at least 5mg, is there any more discount and do you have the same Lot in stock, if not, what’s the lead time?

A:

Yes, we can offer an additional discount for your bulk order. It’s one of our hot-selling products. we don’t have the same Lot in stock, however, we will strictly follow the SOP during the production process of the new batch, and undergo strict quality control to ensure that the inter-batch difference is as small as possible. We currently have many new batches of inventory and we can ship in 3-5 business days after receiving your order.

Q:

As a mAb from ADC drug internalization detection tool, compared with Mab-ZAP, what are the advantages of DT3C?

A:

The advantages of DT3C can be summarized as the following three points:
First, the mAb-DT3C conjugate is simple to prepare, can be formed after only 30 minutes of incubation at room temperature, and can exert the function of the ADC drug administered by the patient in vitro;
Second, DT3C can bind to any IgG from different species (such as human, mouse, rabbit, goat) to form mAb-DT3C conjugates, thus further expanding the extent of DT3C as a detection tool for mAb antibody internalization;
Third, mAb-DT3C conjugates significantly reduced cell viability only when the conjugates were internalized by target cells.
It is worth mentioning that, before DT3C, Mab-ZAP was the only tool to detect the efficiency of mAbs' internalization by cells. Mab-ZAP consists of mouse antibodies and the ribosome-inactivating protein saporin. When mAb-Mab-ZAP conjugates are internalized by cells, cells undergo apoptosis due to toxicity of saporin. Therefore, whether the mAb is internalized by the cells can be detected by the viability of the cells. However, it cannot be excluded that Mab-ZAP itself may reduce mAb internalization efficiency to some extent, since Mab-ZAP is a macromolecule containing anti-mouse antibodies. Compared with Mab-ZAP, the molecular weight of mAb-DT3C conjugate is stable and smaller than mAb-Mab-ZAP. Moreover, related experimental data showed that the internalization efficiency of mAb-DT3C was higher than that of mAb-Mab-ZAP.

Q:

Have you checked the bioactivity of your product Recombinant DT3C (Diphtheria toxin & spg 3C domain) for Antibody Internalization Assay? If yes, could you please share the validation data for biological activity?

A:

Have you checked the bioactivity of your product Recombinant DT3C (Diphtheria toxin & spg 3C domain) for Antibody Internalization Assay? If yes, could you please share the validation data for biological activity?

The related activity testing is still in progress, so we cannot provide the testing results and protocol for customer reference, but we can give some suggestions and references in combination with what we are doing now (literature and product COA).
DT3C uses the 3C domain of Streptococcus protein G to bind the Fc of the antibody. Its binding properties are the same as that of the protein G-binding antibody Fc. It can bind to multiple species of IgG (including human, mouse, rabbit, goat, etc.), and it can also bind to multiple subtypes (IgG1/2/3/4, etc.), but the specific binding strength (affinity) has not been tested.
In theory, one antibody can bind to two DT3Cs (DAR=2), but there may be inhomogeneity, and it may also be related to the specific antibody. The specific value has not been tested for the time being, so it is recommended to explore the optimal coupling ratio in the early stage.
The antibody concentration of 10ug/ml (excessive) can be fixed first, and the DT3C gradient (0-10ug/ml) can be explored to determine the optimal DT3C killing concentration, and then the optimal DT3C concentration can be fixed, and the mAb can be pulled to the gradient to test the internalization efficiency of the antibody. At present, we have received feedback from some customers, most of the testing results are OK, but some are not. The analysis is also related to the cell type. If there is DT3C background killing, it may be that the cell has a non-antibody endocytosis effect, so it is recommended to test a few more cells in the early stage.
The images below are the test result of our DT3C.
DT3C SDS-PAGEDT3C MTT

Q:

What are the binding epitopes of DT3C and antibodies?

A:

DT3C binds to Fc terminal of the antibody.

Q:

What is the specificity of DT3C protein (the impact on cells after addition)?

A:

DT3C utilizes the 3C domain of Streptococcus protein G binding to the Fc fragment of the antibody, and its binding specificity is the same as protein G binding antibody Fc. mAb-DT3C only undergoes killing effects when being internalized into cells through specific binding between antibodies and target cells, DT3C itself has no toxicity to cells outside the cell.

Q:

Could you please provide the complete sequence of your recombinant DT3C protein (CSB-EP360556CQR1)?

A:

Here is the complete sequence including tag sequence, target protein sequence and linker sequence:
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFRT+Target Protein Sequence
GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKGGGGSGGGGSGGGGSIDEILAALPKTDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE)
6xHis: HHHHHH
Thrombin site: LVPRGS
T7 tag: MASMTGGQQMG
Linker: RT

Q:

Can DT3C protein detect the endocytosis efficiency of ADC drug antibodies?

A:

DT3C protein is mainly used to bind with candidate antibodies to simulate ADC drug antibodies, and to detect the endocytosis efficiency of candidate antibodies. However, an ADC drug antibody itself has the function of inducing cell endocytosis, so the endocytosis efficiency of ADC drug antibodies does not require DT3C to assist.

Q:

What is the effective concentration of DT3C?

A:

The effective concentration of DT3C is 1-4ug/ml.
Phase 1: Fix antibody concentration (excessive), pull the DT3C gradient, and determine the optimal DT3C killing concentration.
Phase 2: Fix the optimal DT3C concentration, pull the antibody gradient, and determine the optimal antibody internalization concentration.
Phase 3: The optimal DT3C concentration and optimal antibody concentration will be tested together according to the dilution ratio.

Q:

Please give us your advice about recommended BSL (Biosafety level) for or handling this product recombinant DT3C protein (CSB-EP360556CQR1).

A:

This protein is not contagious or a pathogen. It can be transported normally like regular protein. If a customer just uses this protein for normal experiments or assays. BSL-1 can satisfy the needs. If the customer's experiment is related to the aseptic process. Then BSL-2 is needed. 

Q:

I'm interested in your product Recombinant DT3C (Diphtheria toxin & spg 3C domain) for Antibody Internalization Assay, just wondering if this works on glioblastoma?

A:

We have not conducted any relevant experimental verification yet. If you are interested, we can provide a free sample of a small size for you to test.

Q:

Does incubation of DT3C with antibodies affect their binding if a serum-free medium is present?

A:

You can either use serum-free medium or PBS. Because there is IgG in the serum, IgG will bind to DT3C.

Q:

Does DT3C only have bmk's killing effect?

A:

Under normal circumstances, BMKDT3C undergoes a cytotoxic effect upon endocytosis into cells, while other mAb-DT3Cs also undergo a cytotoxic effect if they can be internalized into cells.
Because when they are internalized into cells, the DT3C is cleaved by cytoplasmic furin protease, the catalytic domain of DT3C released into the cytoplasm. The catalytic domain of DT3C leads to ADP-ribosylation of elongation factor (EF)-2, which subsequently leads to cytotoxicity by inhibiting protein translation machinery.

Q:

Have you conducted DT3C on head to head parallelism experiments?

A:

Sorry, we haven't conducted this experiment.

Q:

How do I know DT3C is linked to the Ab after 30 min incubation with Ab? Will the pH affect DT3C release in the endosome?

A:

Incubation for 30 minute is sufficient for DT3C to link to antibody. We usually suggest that DT3C should be overdosed to reduce the amount of free antibody (antibody that do not link to DT3C) and prevent the free antibody binding target from blocking the binding of antibody DT3C complex.
The pH won't affect DT3C release in the endosome.

Q:

Did you test the toxicity of free DT3C itself to the tumor cells?

A:

This DT3C protein is toxic to the cell only when it is internalized into the cell. We have conducted tumor cell samples before, and each experiment involved a control group with only DT3C. Non internalized DT3C is not toxic to tumor cells.

Q:

How many DT3C proteins are typically attached to a human IgG antibody?

A:

We have not specifically detected the binding ratio of IgG antibody to DT3C protein.
But the 3C domain of DT3C protein can bind to three FC regions, and one antibody (tetramer) has two FC regions. When the ratio of antibody to DT3C is different, the binding will be different.

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