Welcome to today's webinar, brought to you by Promega and Axxam. I'm Kara Machleidt, senior global marketing manager at Promega. I'll be moderating today's event. Before we begin, I'd like to cover a few housekeeping items. You noticed on your console there are multiple engagement windows. All the windows are movable and resizable, so feel free to move them around and get the most out of your desktop space. In this webinar, we have many ways to be interactive. If you have any questions during the webinar, you can submit them through the ask a question box. We're here to answer these questions during the webinar. We'll also save some questions to share during our live Q&A session. Available in the resource library we have additional resources for you to download to learn more. We encourage you to bookmark any links that you feel you may find useful. After the presentation, there is a survey. Feel free to take a moment to answer these short questions. We appreciate your feedback. If you experience any issues with your console, press F5 on your keyboard to refresh your browser. Chrome browser works the best for Windows and Firefox for Mac users. And on demand version of the webinar will be available to you approximately one day after the broadcast and can be accessed using the same web link you received after you registered. Now it's my pleasure to introduce our first speaker, Doctor Christopher Eggers. Dr. Eggers received his PhD in biochemistry and molecular biology from the University of California in San Francisco. And then completed a postdoctoral fellowship at the Howard Hughes Medical Institute at UC San Diego. Since 2011, doctor Eggers has been a senior research scientist at Promega, where he has focused on the development of NanoLuc luciferase and NanoBiT technologies to create new bioluminescence assays that simplifies the measurement of protein dynamics. Now I'll turn it over to Chris. Thank you for that introduction. Today I'd like to introduce the HiBiT protein tagging system and specifically show how HiBiT technology can be used to easily measure both protein secretion and also translocation of membrane proteins to the surface of cells. So HiBiT technology is based on that of NanoLuc luciferase and that. Luciferase was derived from one found in the deep sea shrimp oplophorus. So, in the shrimp it's found as a large tetrameric species. The actual catalytic subunit is small, but on its own it has poor activity and stability. So the advanced technologies group at Promega did a lot of protein engineering to turn that into what we call NanoLuc. And this has a variety of characteristics that make it an excellent reporter and make it extremely versatile in the types of assay formats that we can use. So their next step in engineering NanoLuc luciferase was to turn it into a two subunit complementation system that we call NanoBiT or NanoLuc binary technology. And so to form this, they looked at 91 different splice points in NanoLuc and found that the optimal one here was between residues 156 and 157 because this left just a C-terminal peptide. You could take then the larger subunit of the two and independently optimize this for stability and activity. And that's what we call LgBiT for the for the large subunit. So because the smaller one is just a peptide, we can easily synthesize hundreds of different peptides to look at variants and see how these activate the LgBiT subunit. And we can see that we have peptides that can all activate LgBiT that have over five orders of magnitude of different affinity. So the two bookends here are probably the most interesting, the lowest affinity peptide we call SmBiT. And because this doesn't easily interact with LgBiT, it really only forms the NanoBiT complex readily when brought together in proximity by the binding of two fusion partners. And so this makes an excellent live cell assay for protein:protein interactions. The highest affinity peptide we call HiBiT and this will spontaneously bind to LgBiT with a with a Kd of about one nanomolar. And so this makes an excellent protein tag because we can fuse HiBiT to the protein of interest genetically and then simply add detection reagents that contain both the substrate and saturating amounts of that LgBiT subunit. You only have to wait 10 minutes or less than to generate a luminescent signal, that is then proportional to your the amount of your protein of interest. So because of the brightness and sensitivity of NanoLuc and NanoBiT, we're able to measure extremely small amounts of the tagged protein or like sub-attomole levels, but you also have this very large dynamic range of over seven orders of magnitude. Which means that even highly overexpressed proteins can be quantified in the same assay without having to do any dilutions. So there are essentially three different formats of reagents that contain that LgBiT protein in them. The first is the HiBiT lytic detection reagent which has detergent in the buffer and so will lyse cells and give you a measure of the total amount of HiBiT that's in a sample. The HiBiT extracellular assay does not use any detergent and so the LgBiT will only be able to complement HiBiT that's on the surface of cells or in the media. HiBiT blotting system is very similar to a Western Blot in that you do SDS-PAGE and transfer to a membrane, but it's much easier because you don't have to do any blocking steps or washing steps because you can rely on the specificity of that enzyme complementation event. In that we're making, the LgBiT is interacting with the HiBiT peptide on the surface of the blot and only generating a luminescent signal upon that complementation. A fourth format allows us to look at HiBiT tagged proteins inside of live cells. And this requires expressing the LgBiT protein inside that cell, either through transient or stable transfection or transduction with something like BacMam. What this allows is us to follow in real time changes in the levels of the protein, for instance regulated degradation or PROTAC induced degradation. And we can also then couple it to our NanoBRET technology for measurement of interactions with other molecules, whether those be small molecules or other proteins. You can also multiplex HiBiT detection with a number of other assays to get even more information out of one well. For instance, if you perform a live cell measurement of HiBiT inside the cell, you can do an endpoint measurement of cell titer glow to quantify cell viability or use CellTox Green to look at toxicity of the cells. The HiBiT Nano DLR system allows you to multiplex both firefly and HiBiT in the same assay and this can be great for normalization or to have a control luciferase that you can compare to. And finally the CellTiter-Fluor Assay is a cell viability assay that uses fluorescence. And so it can be paired with any number of luminescent HiBiT detection systems. So in this case, we are measuring live cells because they have a certain protease activity that's only found in live cells. So we can take a cell-permeant fluorogenic substrate that will get into these live cells and give you a fluorescent signal that is proportional to cell number. And this can be measured prior to any of those endpoint HiBiT assays. Just in the last month or so, we have launched a very specific very high affinity monoclonal antibody that recognizes HiBiT and this increases the available assay formats even more, especially allowing immunofluorescent microscopy to show the localization of HiBiT tagged proteins. We can use FACS sorting. We can do traditional Western blotting, which opens up the possibility of doing fluorescent Western Blots or using capillary electrophoresis systems as well. And we can do immunoprecipitation to concentrate and purify HiBiT tagged proteins. So we really think that HiBiT is the ideal tag for studying endogenous proteins and or studying the dynamics of proteins expressed at endogenous levels. It's small size means that it has less effect on the protein function, but it also means that it can be more easily knocked in using genome editing technologies like CRISPR Cas9. We've got simple homogeneous add-and-read formats that allow quantification of proteins in 10 minutes or less, and this can detect very sensitively and as well as across a wide dynamic range, which makes it ideal for measuring even low abundance endogenously expressed proteins. And so, this allows us to measure a number of different types of protein dynamics, not just abundance, but as we'll see later, changes in localization, you know, either internalization or trafficking to the surface or secretion. As I mentioned, HiBiT can be paired very easily with CRISPR technology to add HiBiT at the endogenous locus. Because HiBiT is so small, you can make a template sequence that is just a single stranded DNA oligo that you can synthesize, and this can be paired with purified Cas9 and as a RNP with the guide RNA. So if you electroperate these into your cells, you then in a day or two have simple luminescent assays that can detect the presence of the edit in those cells. So I want to focus now on the HiBiT extracellular detection system. As I mentioned, the LgBiT subunit itself is membrane impermeable. So in the absence of any detergent, it will only be able to bind to HiBiT that's either on the surface of the cell or secreted into the medium. And so any HiBiT that is present on a protein inside the cell will not form any light because it will not be able to bind to LgBiT and so this can give you a simple homogeneous assay for receptor internalization or receptor recycling. Also allow you to easily measure protein secretion or trafficking to the surface of the cell. So one example to show how all of these assays might work together to study these processes is with the protein PCSK9. This is a therapeutic target due to its role in cholesterol metabolism. And interestingly the PCSK9 has a protease domain that is involved in an autoproteolytic processing that occurs in the endoplasmic reticulum and that processing is necessary for its proper secretion. And so there are some well known mutants of this protein that block that secretion. So for instance, H226A is actually knocking out the catalytic histidine of the protease domain that blocks the autoproteolytic processing and therefore causes retention in the ER. But there are other types of mutations like this mutation to C679X that just cause misfolding and therefore cause retention because of that in the ER. So we can compare the HiBiT extracellular and lytic assays to get measures of how much of the protein was secreted and how much of it is still inside the cell. And if we compare that for the wild type protein, we can see that the values are almost identical, meaning that about 100% of the wild type form of the protein is effectively secreted into the medium. But when we look at either of those two mutant forms, you can see that the extracellular signal is much reduced. But when we add detergent to the reagent, we now get a big boost in the signal, showing that in these cases, 1% or less of the protein found in the well was actually secreted into the medium. The vast majority of it was still inside of the cell. And we can look more closely at that by separating out the proteins by SDS-PAGE and doing HiBiT blotting. If we look at just the cells, we can see that both the wild type and the cysteine mutation had active protease that they still had the proper processing of PCSK9 to this smaller form, whereas the histidine mutation knocked out that activity and kept it at the higher molecular weight. But when we look at the media, we can see that only the wild type protein is effectively secreted into the media and both of those other two forms did not. We can follow this over time to see what's happening with the secreted protein by doing an endpoint measurement of either the extracellular or the lytic assays. In this case we've plated cells expressing PCSK9 HiBiT or the mutant forms at time zero and then came in with those two different assays. In this case, we've taken the wild type protein and treated at plus or minus brefeldin A, which can effectively block secretion of the protein as we see by the extracellular signal. So when we look at the lytic signal for total protein, we see that it really had no effect on the production of that protein, just its secretion. Now these were done with a variety of endpoint measurements, but you can also look at the secretion kinetically in real time with live cells by adding both LgBiT and one of our extended substrates to the medium. So if you add endurazine plus LgBiT to the medium, you can follow secretion in real time over 24 hours or more. So PCSK9 was a secreted protein, but we can also look at translocation of membrane proteins to the cell surface. So CFTR is the ion channel, whose when mutated to the deletion of F5O8 is a leading cause of cystic fibrosis. And so the reason why you get cystic fibrosis is because you do not get enough functional CFTR translocating to the surface. And so compounds that stabilize the mutant form of the protein and allow trafficking to the surface can restore partial function and act as therapeutics for that condition. Now we have a little bit of a complication here because both termini of CFTR are cytozolic. So if we want to have an extracellular HiBiT tag, we have to place it internally into a surface loop of the protein. Luckily we went to the literature and somebody else had already done this, inserting an epitope tag, a flag tag in the 4th extracellular loop of CFTR to do their studies. So we just took that same location and inserted the HiBiT tag instead. And as before, we're going to look both at the HiBiT extracellular signal. And also with that same assay but supplemented with digitonin to permeabilize the cell to give us a measurement of surface versus total amount of the protein. So comparison of those two values can can give you a measure of the percent surface expression. And we can see with the wild type protein that again the signals are virtually identical in that nearly 100% of the wild type form of the protein is on the surface. But when we look at that deletion of F508, which we know accumulates in the ER, the extracellular signal is much reduced. But when we add digitonin into the reagent, we see a big boost up in the signal, again showing that the best majority of the protein is stuck inside the cell and is not trafficking to the surface. So we can then treat these cells expressing those two forms of the protein with a titration of two compounds that bind to CFTR and are known to stabilize them, VX-809 and Corr-4A. And so we can see with that titration that the biggest effect that they have is to increase the extracellular signal from the mutant protein. Although there is some stabilization as well of the wild type form of the protein. But if we look at those ratios to measure the percent surface expression, we can see that the mutant form starts at only a couple percent present on the surface and goes up more than tenfold. And so this is why these compounds have therapeutic value, because it can allow the mutant form of this protein to be at the surface at an appreciable level. So in summary, the Nano-Glo HiBiT Extracellular Assay enables homogeneous add-and-read assays that can quantify both secreted proteins and surface expressed HiBiT tagged proteins and this is in 10 minutes or less. So by adding detergent you can change it from measuring only extracellular to measuring total amounts of protein and therefore get a measurement of the percent extracellular in your sample. And this can be performed not only in endpoint format, but by pairing with some of our extended substrates. You can actually measure it kinetically in real time for 24 hours or more. And so really think that HiBiT is enabling HTS compatible quantitative studies of protein trafficking and protein secretion. And that should allow us to better study the regulation of those processes as you'll hear about in the next talk. So thank you very much for attending this presentation. Thank you Chris for a great presentation. Now it's my pleasure to introduce our second speaker, Loredana Redaelli. Loredana has been leading the cell biology group at Axxam since 2007. She oversees their research activities, which focuses on the development and validation of miniaturized, functional cellbased assays for high throughput screening using different reporter systems. Prior to joining Axxam in 2001, Loredana worked as a research scientist at the Bayer Research Center in Milan, generating cellbased assays for high throughput screening for cardiovascular disease targets. Loredana holds a master degree in biological sciences from University of Pavia and a postgraduate diploma in biotechnology from the University of Milan. Without further ado, I will turn it over to Loredana. Hello, Thank you for the introduction. My name is Loredana Redaelli, and now I'm going to talk about the applications of HiBiT technology in protein secretion, trafficking and regulation. So what about the topics of this presentation? At the beginning, I will show you some slides about the secretory pathways, so some background cell signaling and some potential therapeutic application. Then I will share with you some examples using HiBiT technology on protein secretion and trafficking and finally the summary of this webinar. So let's start with the first part, some background on the secretary pathway. So, as you know, the human proteome has about 20,000 protein coding genes, but what about the secreted proteins? So what we know based on the prediction of the signal peptides about 15% code for secreted proteins. But I mean, for sure, I mean this is an estimation and this really depends on the age of the cell on the cell types. So in particular if you take a look of the fraction of transcripts in some tissue like pancreas and salivary glands, we can also reach 70%. Now moving to the secretory pathway on the right, we have a general schema about the conventional protein secretion in eukaryote. In what we know, what we know from literature is that the secretory pathway has protein delivered to different membranes such as ER, golgi, lysosome and plasma membrane. The secretory pathway has also protein delivered to intracellular compartment as well as secreted protein delivered to the extracellular space. Moving to the say signaling what we know from from literature is that for sure protein secretion is a fundamental cellular mechanism by which either soluble protein or membrane protein are released into the extracellular space. In particular in order to respond to changes in the space, but also in order to respond to signal from other cells. So overall I mean we can say that the protein secretion influences different biological function and in particular concerning secreted proteins and the receptor, they are responsible for let's say communication. And so, if we can take a look on this diagram, I mean they are responsible for communication between cells, between tissue and also organs in our body. The key message that I would like to give you here is that for sure the regulation of these pathways is important for normal human homeostasis. So in case of this regulation for sure we will have an important impact in pathophysiology and disease. So based on that, moving to potential therapeutic application, we know, from literature, again, this is let's say a schematic representation of the mammalian protein secretory parthway. We know that based on literature, there are molecules acting at different steps and at different points. In particular, there are some molecules acting at the ER import, others at the level of protein maturation, others working at the level of trafficking. And so overall, I would say that the key message here is that the possibility to identify molecules regulating the secretion of protein of interest for sure, can offer, can represent let's say an important tool for diverse diverse range of function in both prokaryotes and eukaryotes. And so this molecule can have an important therapeutic applications. Before moving to the, let's say example, I would like to to share with you a general, I would say schema focusing on the operational activities at Axxam. So Axxam is an innovative partner research organization. And it is mainly focus on the early steps of the drug discovery. If we can take a look on the other part of this slide, I mean this is a schematic representation of the different steps of our operational activities. And in particular, I would like to focus your attention on the technology implementation, because we can develop innovative assay in order to monitor important cellular processes such as protein:protein interaction, protein degradation, and protein secretion. We can analyze different classes of targets and so based on the target we can let's say we have the dedicated team, so in case of an enzyme or let's say a cell free system, we have the biochemistry team, I mean the cell biology. Later we will have a dedicated focus on this. In case of electrogenic protein we have the electrophysiology. In case of phenotypic screening, cell painting the High Content team. In case the interest in is in relevant cellular model, we have the IPS. And then let's say the lead discovery department concerning high throughput screening, compound management, data science, analytical service with Med Chem support. So now focusing on the cell-based assay development process. I mean this is let's say a general schema of our activities starting from target nomination down to HTS. For sure at the beginning we have I mean as soon as a target is being nominated, we in collaboration with our molecular biology group, we generate the molecular biology tools then we select the most suitable cell line and read out. We typically perform a stable transfection and limiting dilution in order to purify single clones. And here we have let's say the imaging of the instrument that we use and we typically do functional clone selection. That means comparing target prospective cells versus negative control cells. And during the optimization we fully characterize the clones and then before moving to HTS, we validate the GSA testing different quality criteria. So now moving the to the example, the first one is about a cellular enzyme secretion. So here the goal is to generate an assay in order to identify secretion enhancing molecule. So in other words our goal is to reduce the ER aggregation of the mutant isoform and in order to increase the extracellular level of the mutant isoform. What about the assay type? We have generated two different cell line, two recombinant cell line. So one expressing the wild type, the other one expressing the mutant isoform of the target. And the target was labelled with two let's say reporters, one the HiBiT and the other one is the GFP. So just have a look on the schema on the right. So as I said before. In orange the wild type in the violet the mutant and then label with both GFP and the HiBiT tool. In let's say normal condition, in wild type cells, the protein is secreted in the medium. But what about the mutant? The mutant is retained in the ER. And so our goal is that in the presence of a modulator, we will in some way restore the, let's say, physiological condition and we will have an increase of the extracellular level of the enzyme. Moving to the assay principle, we have essentially developed two assay. The first one is an HiBiT based NanoLuc complementation assay using the extracellular let's say detection system on the right that was previously described. And so here we let's say measure luminescence signal in order to analyze the extracellular protein amount. While the other two assay that were fluorescent based assay in this case, I mean we analyze the intracellular protein amount both using a fluorescent plate reader, but also we perform an imaging base assay with the main goal to determine protein localization of the target of interest. And so, focusing on this assay, on this imaging base assay, now I will show you some data using this assay. First, I would like to underline that we use an inducible expression system and we have generated mammalian stable clones. And concerning the images were acquired at operator using two channels, bright field and GFP. And so here we have the data compared in the absence and in the presence of the inducer. And as you can see here, I mean we obtain an increase in the fluorescence intensity. And several ER aggregation in self expressing the mutant isoform. Now moving to the hybrid, I say in this case also in this case we compare wild type versus mutant and due to the fact that this is an usable system, we perform let's say doxycycline dose response curve. And here we have the luminescence data, and what we obtain is that the HiBiT tagged protein is more secreted in wild type enzyme compared to the mutant ones. Moreover, we also tested I would say a generic tool like 4PBA and here we have the data on the mutant isoform and what we got is that in the presence of 10 millimolar we observe an increase of the luminescence signal. Now moving to an additional experiments, we perform a further characterization on this cellular enzyme secretion assay, in particular comparing extracellular versus total I would say the lytic protein. Concerning the extracellular, here we have the data in the presence of one concentration of doxycycline comparing wild type versus mutant. And also here we also that the wild type was abundantly secreted in the presence of the inducer while low level low secretion in the mutant one. Moving to the total, we use a deleted protocol that was previously described. In this case, I mean we got similar signals, so similar total amount in the presence of doxycycline in both wild type and mutant isoform. So now moving to the other example, in this case this is about a plasma membrane protein and here the focus is the trafficking. So the goal is to develop, to generate an assay in order to identify trafficking modulators of these of these protein. And in particular in order to enhance its membrane localization in particular of the mutant one. Concerning the assay, we have generated recombinant cell line expressing either the wild type or the mutant isoform or the target. And in this case the target were labeled with only HiBiT. So a little bit different compared to the previous example. Concerning the assay principle, we have developed two assays, the first one is about HiBiT based NanoLuc complementation assay. I mean. Here we have the schema of the intracellular lytic, and in this case I mean was let's say a luminescence signal recording for let's say total protein amount. And here we have the data for only the mutant one in the presence of increasing concentration of the compound. While concerning the other assay, in this case was an imaging immunofluorescence. We have used the Anti- HiBiT antibody and in this case the main focus was the protein localization analysis and here we have some, let's say representative analysis in the presence of two concentrations of the compounds. And on the right, on the right some let's say HiBiT intensity related again with in the presence of increasing concentration of the compounds related to plasma membrane. And I mean both the two data are in line let's say. Now moving to, let's say the optimization part that we generally do during, let's say before, let's say performing an HTS. So in this phase we generally verify different parameters in order to define the best assay protocol. And here this is let's say a general experimental workflow. So from cell seeding, cell treatment, cell incubation and then injection of the Nano-Glo HiBiT Extracellular Detection System and finally luminescent measurement. So I mean with different instruments that we have available for sure. I mean this is a very straightforward protocol. So a live and homogeneous protocol using the specific cell impermeable HiBiT Extracellular Detection System. And before moving to the HTS we typically have to verify different quality parameters in particular RZ' factor. That should be higher than .5 and intra- inter- plate coefficient of variation lower than 20%. And for sure the pharmacology of the the reference compounds, so we needed to obtain a reproducible reference compound AC50. Moving to this slide, this is an example of our multiplate test using related to luminescence extracellular detection. In this case the the plates were tested using, as I said before the final optimize protocol. And here we have the data regarding assay robustness that was proved through a multiplate test, in particular concerning the Z-prime factor and rZ' prime factor were higher than .5. And also a good performance was also obtained for the coefficient of variation intraplate and interplate because in both cases we obtain a value lower than 20%. Concerning the CR means the central reference, that means the neutral control, while the CR means the scale reference. So in the presence of the the reference compound. Also concerning the central area, I mean the value were very good. And due to the fact that the time of incubation was long, we also perform a viability assay. In particular, we use the Cell Titer-Fluor Cell Viability Assay. So we perform a multiplexing experiment. So first we we measure fluorescence and then the luminescence extracellular detection. And before moving to the conclusion, I would like just to show you this, I would say very, very general workflow mainly related to HTS that we typically perform during a screening campaign. So with the different, with the different steps. The first step is the assay transfer, that means that could be related to assay developer provided by the client. Then we have the adaptation to automation of the HTS assay. In this case, we have them, we run multiplate test, we define the full automation protocol. We evaluate assay performance for both primary and interference assay. An important step is the pilot screening. We typically test three to ten thousand compounds over two days. And the goal is to confirm the robustness of the assay, to define the screening protocol and to the define the data analysis, the the data analysis method and to identify the concentration of the compound to be used during the primary screening. Typically on one assay while it confirmation again one concentration on both primary assay and the interference one, and then the the potency determination. In this case a complete dose response curve of the positive hits. And finally they follow up activities that means orthogonal assay and selectivities and so on. So, going to the conclusion of this presentation, I mean this is the summary. So during this presentation we talk about the application of the HiBiT tool in particular in protein secretion and trafficking field. We generate the different HTS compatible cellular assay rely on the HiBiT system and I mean for sure these live and homogeneous cell protocol is I would say convenient for running HTS campaign using the specific cell impermeable Nano-Glo HiBiT test a cell detection system. So this is all from my side and thank you for your attention. Thank you, Loredana for a wonderful presentation. Now I think we'd like to start our live Q&A session. Just a reminder again and you can submit your questions in the answer question box. Looks like we have a number of questions already coming in during the presentation. I'll start with those. There's a OK, one question. When looking at protein degradation, what are the main advantages of HiBiT over the more traditional immunoassays such as HTRF for example? Yes, Chris, yeah, I could answer that. So one thing that we have found when looking at regulated or induced protein degradation is that it's very important that the proteins be expressed at endogenous levels because otherwise you can more easily throw off the stoichiometry with the degradation machinery. And that's why knocking in HiBiT using CRISPR is such a powerful technique for studying those. So you may ask why not just use a an immunoassay and just study the you know non-tagged protein? I think that the main advantage over immunoassays would be for HiBiT just the very large dynamic range. So we've got the sensitivity to sub-attomole levels you know so you can quantify even weekly expressed proteins in cells all the way up to highly overexpressed proteins. So that's all in the same you know greater than 7 log linear dynamic range which means you don't have to dilute samples if they're too high and you still have the sensitivity. And then you know secondly would just be the ease of use that it's just an add-and-read assay, you add the reagent and 10 minutes later you have the the luminescence. So from the workflow it's easier there. And then you're not relying on, you know, needing to have a very high affinity specificity antibody against your protein of interest, which can be hard to find. And so you know, you may not have a good antibody to allow you to very sensitively detect the proteins at their endogenous levels. Great. Thanks, Chris. Another question for lysing HiBiT cells, does it require the use of protease, this protease blocker to ensure optimal signal? Yeah, we didn't find it necessary to add protease inhibitors. So the if you're using the HiBiT lytic reagent, just the buffer conditions there produced, you know a signal half-life, you know in excess of two hours and that was kind of whether you were just using purified protein or using cells. So we certainly didn't see any signs that degradation of the protein was causing a decrease in the signal over time. You're more than welcome to you know try it out and add and and see if that would be helpful. But it wasn't something that seemed to be necessary for the HiBiT lytic assay. Thanks Chris, maybe this one for Loredana, do you have some experience with the genome editing approaches using the HiBiT technology also for high throughput purposes? Yeah, I mean we have let's say very good experience with genome editing approaches. So typically let's say based on the final goal of the project, based on the target we have used different cell line and at the beginning of the project we typically made some let's say preliminary activities in order to verify for instance, the level of the expression of the target of interest. So we generally perform some qPCR analysis. Moreover, we typically do some sequence analysis at the level of the loci before doing the insertion of HiBiT. And again, another important step is to verify the suitability of the cell line for HTS. So we verify the cell growth and so on. All right. Another question for Loredana perhaps. Did you use different cell types applying the HiBiT technology? Yeah, I mean, along this year we have tested, let's say, different cell types again based on the final goal of the project. For sure we have used the classical HTS, let's say cell line like HK cell line, but also for instance different cancer cell line, again adherence cells in suspension cell. So I mean different cell types. Thanks. I think this one's for Chris. For the CFTR study, you inserted HiBiT into internal loop of the protein. Are there any special considerations when using this approach versus terminal tagging? Yeah, so the in order to form an active NanoBiT complex or NanoBiT luciferase, the HiBiT peptide actually has to adopt a kind of extended beta strand confirmation just to fit into that structure, and so it does need some amount of flexibility to to give you the maximal signal there. So if you do insert HiBiT internally into a tight loop on a, on the surface of a protein, just because it's, you know, bending it, you can see a reduced signal. So in the CFTR example for instance, we started with just a four residue Gly/Ser linker on either side of HiBiT and we did notice you know significantly reduced luminescent signal compared to if HiBiT were just on the terminus where it's fully accessible. You know still light that you could measure but it was reduced from kind of the maximal level and so as we added as we increase the linker size on either side the the signal went up substantially in this case. So for the data I showed, I believe it was a 12 residue Gly/Ser linker on either side of HiBiT in that loop, just to make sure that it had maximal flexibility for binding to the LgBiT. So you know that may or may not be necessary. It kind of depends on how much signal you need and what the expression level of your protein is, but that that can be important to get full signal from the HiBiT. All right. In the interest of time, I think we can address one question. These two questions are similar. So maybe both of you can kind of, so basically how important it is for these studies to use endogenous expressed proteins from CRISPR modified cells rather than transfected expression construct or that's another one, any experience of transient transfaction? So all the, so I mentioned kind of in the previous question the importance of endogenous expression when looking at regulated protein degradation and that's I think just because of the stoichiometry to the degradation machinery is very important. So in in the studies I showed today that was all transiently transfected expression constructs and you know, so I think it will really depend on just. You know for your particular protein or process whether you know there's an overexpression effect on that. For the the proteins I showed, I was, you know, mainly looking at, you know, mutant forms of that that were getting stuck in the ER and not properly trafficked. And you know, it did not seem that just using transient transfection was a problem at all for for measuring those. I should say that we were just isolating a CRISPR clone of PCSK9 HiBiT and so hopefully in the coming months I can you know compare the two and see if there is an important difference there. But I think for many systems you will be able to do either. It's really just a question of whether you know there are whether there's an overexpression effect caused by the need to interact with other proteins at specific stoichiometries. So here we haven't seen that yet, but we haven't done a whole lot of studies on regulated secretion with CRISPR cell lines yet. Alright, thanks Loredana, do you want, do you have any comments on that? Yeah, just only find that comment from my side, I mean. What we typically do during the assay development, we use transient transfection perhaps at the beginning of the project to choose the cellular background and also to identify the best assay protocol and then what we typically do a stable transfection as indicated in the example that I have shared. Thank you. Thank you both of you. We still have some questions that I haven't got to answer, but in the interest of time, we'll have to stop here. We will follow up with those questions via e-mail. 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