The Protein Interactions Expert



FAQ

General

Who will be my contact at Hybrigenics?

You will be in contact with both a sales engineer having scientific and technical skills and a PhD scientist.

Your sales engineer will guide you in the choice of the best service for your project, will send you a quote and help you with the administrative aspects of your order.

Your dedicated scientific project leader will discuss in details about your research, your project needs and will advise you on the best technical strategy for your project. They will follow up on the progress of your project and will be available to review your results.

If you want to discuss with our team, you can contact us now.

Can I get a confidentiality agreement before discussing my project?

Yes, Hybrigenics can provide you with a template of a confidentiality agreement, to be reviewed and adapted to the specifications of your project. You can also propose your own template.

Please note that any project discussion and project results are kept confidential

Logistics

What material/sample do I need to provide?

For most of the services we offer we would need to get a DNA template: ULTImate Y2H , ULTImate Y2H+1 , ULTImate Y1H , Loss of Affinity Mutant Screen , 1-by-1 Y2H & Interaction Domain Mapping , 1-by-1 with HTRF® , 1-by-1 with Biacore® .

We recommend sending 5µg of purified DNA of kit purified DNA at 200-400ng/µl in water or Tris (with no EDTA) or as a dry pellet (not on a filter paper).

For ULTImate YChemH projects, we handle the synthesis of your derivatized molecule. You don’t need to provide compounds.

For ULTImate screening projects, we use our own libraries as they are optimized for our process so you don’t need to send a cDNA library. Just choose one from our list. However, we can prepare a custom cDNA library from RNA samples you provide.

In that specific case, we generally ask to receive 1 mg of total RNA, as we expect a yield of 1% to 5% polyA+ RNA after extraction. With 10 to 50 µg of polyA+ RNA, we can attempt several shots of cDNA synthesis if needed.

For specific projects with in vitro interaction experiments, HTS compound library screening or hit to lead services, you may need to send protein samples or compounds in well plates. Contact us for more details .

How should I send my sample?

If you are sending us DNA samples, you can simply send them in tubes with Parafilm by normal ground mail. They don’t need to arrive quickly as DNA is stable.

If you are sending us RNA or protein samples, please use a large package filled with a lot of dry ice and ship it by express mail. This type of material is very fragile and must be kept at least at -20°C. We recommend that you don’t send your parcel before a week-end to make sure that we will be present to immediately store the samples at the appropriate temperature.

If you are sending a small number of compounds; you can ship powders at room temperature by normal ground mail. If we need to receive many compounds, we will require compounds in DMSO in 96 well plates on dry ice (Micronic or equivalent).

Where should I send my sample?

Please address your template to our labs at the following address:

Hybrigenics Services
3-5 Impasse Reille 75014 Paris
France

Ultimate screen process

How do you test bait toxicity?

First, the plasmid containing your bait is transformed in haploid yeast cells with mating type a. The bait plasmid enables yeasts to grow on selective medium lacking Tryptophan.

In order to check if the bait is toxic in yeast, we spread the yeast cells transformed with the bait plasmid on two different media: a rich medium (no need to keep the bait plasmid for growth), and a selective medium lacking Tryptophan. The bait protein is constitutively expressed and if it is toxic to yeast, the yeast cells either reject the corresponding plasmid, therefore losing their ability to grow on selective medium lacking Tryptophan or keep the plasmid but the bait expression is toxic and thus the yeast cannot grow. We then compare the number of clones growing on both media and calculate a toxicity ratio between the selective medium lacking Tryptophan and the rich medium.

If your bait is too toxic for our yeast strain, we will transfer it into an inducible vector to allow its expression just for the mating reaction.

Can you explain to me how the test screen is performed?

The test screen is performed to determine the optimal conditions for the final screen. We evaluate mating efficiency and choose selection pressure.

Our prey libraries are pre-transformed in a yeast strain with the mating type α. They contain 10 million independent clones. The prey plasmid has a Leucine selective marker. The bait plasmid, containing a Tryptophan selective marker, is transformed in a yeast strain with a mating type a. A cell-to cell mating is then performed to form diploid cells containing a bait and a prey plasmids. This process is much more efficient than co-transformation of two plasmids in yeast. We evaluate the mating efficiency (number of interactions that can be tested) by spreading a dilution of the mating reaction on selective medium lacking Tryptophan and Leucine to select for the presence of both bait and prey plasmids. We check that we will test a minimum of 50 million interactions, corresponding to 5-times the complexity of the library.

Also, a defined amount of the mating reaction is spread on several selective media without Tryptophan, Leucine and Histidine, the selective marker for the bait and prey interaction, but increasing concentrations of 3-Aminotriazole (3-AT). 3-AT is a competitive inhibitor of the HIS3 reporter gene product and is used to increase the selection pressure and as a consequence to reduce background noise. The optimal condition for the screening is the one with a minimal background growth and a maximum number of positive clones. The lowest possible selection pressure is chosen for the final screen.

What kind of bait can I screen against a cDNA library?

Our ULTImate screening process has originally been developed for the discovery of protein-protein interactions in the Yeast Two-Hybrid (Y2H) system (see our ULTImate Y2H offer). The bait protein can either be soluble or membrane-bound.

In the specific case where you are looking for membrane protein partners, we propose another Y2H technology based on the split-ubiquitin system (see MBmate Y2H ).

By the way, if your bait protein requires a cofactor or a post-translational modification, we can express an additional protein as part of our ULTImate Y2H+1 service.

We have also adapted the system and our process to the screening of:

  • DNA-protein interactions . DNA sequences, like promoter regions or responsive elements can be used as baits once they have been integrated to the yeast genome. Learn more on ULTImate Y1H .
  • RNA-protein interactions . You can find RNA Binding Proteins and study gene expression regulation. Discover ULTImate RNA Y3H .
  • Small molecule-protein interactions . It is also possible to identify the protein targets and off-targets of a small molecule, in the context of a drug discovery and development project. Find more info about ULTImate YChemH .

Libraries

Can I buy Hybrigenics cDNA libraries?

No, we do not offer our libraries to other companies or academic institutions for in-house purposes. Hybrigenics’ domain-enriched high complexity libraries are proprietary and have been uniquely constructed to be used with our ULTImate screening process. By the way, it is necessary to have a very exhaustive screening protocol to be able to reach saturation that we offer in our ULTImate screening services.

I don’t find the appropriate library in your list; can you prepare a new one?

Hybrigenics offers more than 100 libraries from human, rodent, plant, bacteria and other model organisms that cover many research areas.

However, if you wish to screen your bait with a specific library that we don’t have available, we propose a library construction service and can prepared a custom one from your RNA samples. See What material/sample do I need to provide? In the LOGISTICS section.

Ultimate screen results & deliverables

What happens if no/few positive clone(s) is/are growing?

In our ULTImate screening offer, we are screening your bait in a first vector and freely perform a second screen in a second vector with a higher sensitivity in case we obtain less than 20 positive clones.

What happens if my bait cannot be screened?

We investigate the feasibility of the screening project by testing the bait toxicity and self-activation. If despite of a lower expression of the bait or the addition of a signal inhibitor, the required screening conditions cannot be reached, we will stop your screening project.

In that very rare case, we will charge you only 30% of the total fee of the project. Your scientific project leader will advise you on another strategy, should you wish to start another project with a different bait.

What are the deliverables I will receive?

Deliverables consist mainly in results files with both raw data and analyzed results.

They contain a summary of the experiments performed, an analysis on interaction domains and a ranking of the interactions thanks to Hybrigenics confidence score. For more details on our deliverables, visit this page .

Upon request, you can also receive up to 5 prey clones for no additional fee. If you want to obtain more positive clones from your screen, they are available to buy. You will receive extracted cDNA as mini-prep tubes after signing a simple Material Transfer Agreement.

What is the average time-line?

Once we have your approval on the project strategy and your sample, the project time-line is 3 to 4 months on average.

What is the price?

Price depends on the project that will best suit your needs. You will need to discuss with a technical sales engineer to get an evaluation and a quote.

Invoicing

When should I pay for the service I ordered?

Except for specific and customized services in the INHIBIT line, the invoicing is done at the results delivery. Invoices shall be payable net thirty (30) calendar days from the invoice date.

If your administration is requiring anticipated or splitted invoices, we can adapt.

What currency do you accept?

We can receive payment both in Euro (€) or in US Dollar ($) from labs based in the US or Canada.

Publication

Will Hybrigenics retain intellectual property on my results?

The results resulting from Hybrigenics experiments for its customer are the sole property of the customer who is therefore free to communicate and publish the results.

Hybrigenics only uses the results internally for statistical purposes and will never disclose it.

All related publications shall mention Hybrigenics contribution according to standards and custom.

If you can’t find the answer to your question, contact our team . We will be happy to provide you with detailed information.

We also invite you to browse our section HGX & YOU .

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