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Review the advantages and limitations of different strategies applied for the production and testing of anti-GluR antibodies with Professor Elek Molnár.
Elek Molnár graduated in General Medicine (MD) from the Albert Szent Györgyi University, Szeged, Hungary in 1986, where he remained for some time as a Research Fellow in Biochemistry. He then received his PhD at State University of New York, Syracuse, USA.
In 1991, Elek joined the Medical Research Council (MRC) Anatomical Neuropharmacology Unit, at the University of Oxford, UK where he started his studies of GluRs. In 1997, he was appointed as Lecturer in Pharmacology at the University of Newcastle upon Tyne, UK.
In 1999, Elek joined the MRC Centre for Synaptic Plasticity, University of Bristol, UK and in 2004, was appointed as Professor of Neuroscience.
Elek studied the developmental and activity dependent changes in the molecular organization, signaling and function of GluRs. He used a combination of molecular, pharmacological, immunochemical and imaging approaches to gain understanding of the roles of GluRs in the central nervous system.
Hello, and welcome to Abcam's webinar on Investigation of glutamate receptors using immunochemical techniques. Today's principal speaker is Elek Molnár from the University of Bristol in the UK. Elek graduated in General Medicine from the Albert Szent-Györgyi University in Hungary in 1986 and remained there as a Research Fellow in Biochemistry, until he moved in 1989 to the State University of New York. This is where he carried out research for his PhD. In 1991, Elek joined the Medical Research Council at the University of Oxford where he started his studies of glutamate receptors. In 1997, he was appointed as Lecturer in Pharmacology at the University of Newcastle upon Tyne, and in 1999 Elek joined University of Bristol. In 2004, he was appointed the Professor of Neuroscience at the university. He has studied the developmental activity dependent changes in the molecular organization, signaling and function of glutamate receptors. Elek used a combination of molecular, pharmacological, immunochemical and imaging approaches to gain understanding of the roles of glutamate receptors in the central nervous system.
Joining Elek today will be Elizabeth Day from Abcam. Elizabeth has a PhD in Molecular Biology from the University of Bristol, and she has worked at Abcam since 2012 within the small molecule inhibitors team contributing to the growth and development of the catalogue. Before we start, I'd like to just give you a quick reminder that questions can be submitted at any time via the Q&A panel on the right hand side of your screen. I will now handover to Elek who will start this webinar.
EM: Thank you, Lucy, for the introduction. As most of you know, glutamate receptors are the major exciter in neurotransmitter receptors in the central nervous system, they are involved in the very wide range of signaling events. Over the last 24 years, several antibodies were developed against various glutamate receptor proteins, and these are extensively used in a number of studies. So in today's webinar I'm trying to highlight a couple of key issues that you need to consider when you start to use some of these immunoreagents. So I will start with a brief introduction to the molecular organization and regulation of glutamate receptors, and I will highlight some of the special challenges associated with the investigation of these receptors. Then I will move on and overview some of the strategies used for the production of these antibodies, their advantages and limitations. Then I will overview some of the immunochemical approaches used for the investigation of glutamate receptors, with special emphasis on the validation of antibodies, and the required controls for various immunochemicals that mix. We will also discuss common problems, pitfalls in execution of these techniques or interpretation of data.
So, first, a brief historical overview. As you all know, glutamate is a simple amino acid. Identification of glutamate as an exciter in neurotransmitter in the central nervous system emerged slowly in the 1950s and 1960s. Early studies identified a high level of glutamate throughout the central nervous system. If it's applied locally the glutamate produced convulsions, and also excited single neurons.
In the 1970s, pharmacological tools identified various subgroups of inotropic glutamate receptors, which responded preferentially to AMPA, kainate and NMDA receptors. These are all agonists of different subsets of inotropic glutamate receptors. These pharmacological tools revealed a considerable function and diversity for these receptors, and also the role for glutamate receptors in the neural degeneration and neuronal cell death was identified.
In the mid-1980s another type of glutamate receptor was identified, which are not inotropic ligand-gated ion channels like the previously known receptors, these are G-protein-coupled and they operate through secondary messengers.
Inotropic and metabotropic glutamate receptors remained fairly aloof even until the late-1980s/early-1990s, when expression cloning identified some of the key members of this receptor family. Soon after the first sequences started to appear, homology cloning identified a really complex family of glutamate receptors, both inotropic receptors with several subunits, metabotropic glutamate receptors with various isoforms. Each of these various splice variants, and some of the inotropic glutamate receptors had subunits at various editing sites. The availability of these new genes and sequences that facilitated the site-directed mutagenesis studies which identified key or minor site residues and key domains involved, for example, in ligand binding or channel pore formation, or conformational changes, and these studies mapped the hotspots in the sequences. In parallel, various transgenic animals were developed and these allowed the functional characterization of these proteins in behaving animals.
In recent years, several new pharmacological tools appeared and these efforts that were facilitated by the availability of various x-ray crystal structures. Initially, just the ligand binding side, but more recently the entire subunit structure was identified using this technique. This led to various molecular modelling studies and that fed into the rational drug design side, and using this information helped researchers to develop higher affinity and more cell-active pharmacological tools. Also, clinical trials with memantine for the treatment of Alzheimer's Disease generated considerable interest. The first anti-glutamate receptor antibodies appeared in 1990. These antibodies were used for various localization studies, both at light and electron microscopy level, and also helped us understand the molecular mechanism of glutamate receptor signaling, provided vital information about the composition of the native receptors, also helped us to understand phosphorylation; the phosphorylation events. Identify the very wide range of protein-protein interactions of associated proteins, and also helped us to establish the rules of receptor trafficking to the cell surface and targeting to the various synapses.
More recently, antibodies are used to retrieve the native receptor complex and in combination with proteomics, we can now analyze the changes in the composition of native glutamate receptors.
So this slide shows the family of inotropic glutamate receptor pore-forming domains. This is a sequence dendrogram where based on the sequence homology, and these subunits are grouped together into four different groups: AMPA, kainate and NMDA preferring inotropic glutamate receptor subunits. There is also a fourth group, these delta (δ) subunits, but we know very little about them because they don't seem to form functional channels, and they don't seem to respond to endogenous agonists. Within these groups, these subunits tend to form functional channels in various combinations, and the number of different splice variants are available for various subunits.
So all of these inotropic glutamate receptor subunits share a common basic structure membrane topology, they all have a large extracellular end terminal domain. This region, which precedes the first transmembrane domain called S1 domain, together with this S2 domain, which is looped between the last two transmembrane domains forms the ligand-binding site. Each subunit carries its own ligand-binding site, and there are three transmembrane domains, and there is also a hairpin light structure which forms the pore of the ion channel, and this region shows a resemblance to voltage-gated potassium channel pore-forming domain. The sequence of this region determines the ion cell activity of the channel. Also, there is intracellular C-terminal domain which has various phosphorylation sites, and further a protein-protein interaction site. While at the C-terminal domain, most of these receptors are linked to scaffolding proteins, or the cytoskeletal. Some of the inotropic glutamate receptor subunits also have cleavage sites for the calcium-sensitive endopeptidase. So this is one subunit, four of these need to assemble to form a functional ion channel.
So different types of inotropic glutamate receptors have different roles in synaptic transmission. AMPA receptors are responsible for fast synaptic transmission, they are the key components of the modifiable synaptic response, which can be enhanced and reduced depending on the requirements. Kainate receptors, they regulate the activity of neuronal networks, they are present both pre- and postsynaptically. Presynaptically they modulate neurotransmitter release, both at glutamatergic and GABAergic synapses. Postsynaptically they contribute to the slow component of the excitatory postsynaptic current, and they also inhibit potassium channels through a metabotropic action. NMDA receptors are partly recognized modulators of synaptic response, they can not only respond to glutamate receptors, but they can also detect vaporization of postsynaptic membrane. This vaporization is normally carried out by other inotropic glutamate receptors. So they can read the pattern of synaptic activity, and based on that they can trigger intracellular events which are the basis of synaptic plasticity, such as long-term potentiation and long-term depression of synaptic communication which can lead to enhancement and reduction of synaptic coupling between neurons. These simple cellular processes seem to underline various types of learning and memory.
Here, this sequence homology dendrogram shows the metabotropic glutamate receptor family. Based on the sequence homology, pharmacological properties and coupling the intracellular signaling pathways, they are divided into three groups. The first group contains the mGlu1 and 5 isoforms of metabotropic glutamate receptors. These are linked to the activation of phospholipase C (PLC), which leads to an increase in the inositol trisphosphate (IP3) and diacylglycerol (DAG) synthesis. Group two and three both inhibits adenylyl cyclase (AC) enzymes, and that will reduce intracellular cyclic adenosine monophosphate (cAMP) levels.
Metabotropic glutamate receptors like other G protein-coupled receptors have seven transmembrane domains. They have a large extracellular end terminal domain, and this is also called the Venus flytrap domain. Binding to glutamate to this domain will lead to the closure of this Venus flytrap domain, which will induce changes, structural changes, in the transmembrane domain, and that will influence the binding of the receptor to the corresponding G protein.
Metabotropic glutamate receptors have a very diverse role in synaptic communication, they mediate slow excitatory and inhibitory responses. They also regulate calcium, potassium and non-selective cation channels. They can also inhibit or facilitate neurotransmitter release, and they, like NMDA receptors, play a prominent role in the induction of long-term potentiation and long-term depression. They also contribute to the formation of various types of memory. They have an effect on inotropic glutamate receptor trafficking, for example, on AMPA receptor internalization from the cell surface. They can also modify NMDA receptor mediated synaptic transmission. They have an important role throughout the development of the central nervous system, and because they are present in glial cells they are involved in the communication between neurons and glial cells.
In addition to these pore-forming subunits and various metabotrope isoforms, there are a number of glutamate receptor associated proteins. They fall into two categories: one is the relatively more recently identified auxiliary subunit group. There are four key criteria based on which we can classify an interaction partner as auxiliary subunit. First of all, these proteins are not integral components of the channel pore, they interact directly and more or less permanently with the pore-forming subunits, and they tend to influence multiple aspects of receptor function, pharmacological properties, subcellular trafficking and targeting. Usually, the assembly of these auxiliary subunits is required to form the receptors which resemble the native in vivo receptor complex. Without that there is not going to be a perfect match between the recombinant receptor and the native receptor, or what you can find in the brain.
There is a very large number of other interaction partners, usually these interactions are transient, often fairly dynamic, and usually these have a more targeted effect on the receptor function. They influence singular aspects of receptor function like biogenesis, trafficking or synaptic localization.
Here, on this table, I've collected some examples for these for various glutamate receptor types. I highlighted in red the auxiliary subunits, which permanently interact with these receptors directly, and there are a number of examples for transient interaction partners. If you quickly scan through this list you can see that there are some overlaps, but generally a unique set of these proteins interact with any subset of glutamate receptors.
Glutamate receptors play an important role in the healthy brain, but they are also involved in a number of neurological and psychiatric disorders, like several of the neurodegenerative disorders: epilepsy, anxiety, stress disorder, depression, schizophrenia, fragile X mental retardation, Parkinson's Disease, chronic pain, migraine, drug addiction, hypoxic brain damage and excitotoxicity, which is basically the over-excitation, over-stimulation of these receptors are responsible for neuronal cell death in many of the neurological disorders.
So now changing topic and looking at antibodies, I thought before I discuss specific glutamate receptor-related issues, I'd just remind you about the key principles of antibody antigen interactions. So these interactions are highly specific between the antibody and antigens, and these antibodies can recognize relatively small regions of antigens. That's a major advantage for glutamate receptors, because, if you remember, these show very high sequence homology and only small stretches of the sequence are unique. It's ideal for discrimination between highly similar proteins such as glutamate receptor subunits or isoforms. Antibodies bind to complementary antigens by three-dimensional recognition, and the antibody antigen complex is stabilized by non-covalent bands, and the binding of antibodies to antigens is reversible and the interaction will form the equilibrium reaction.
So this is a reminder of what an IgG complex looks like, there are two heavy chains, two light chains linked together by disulfide bridges. This light blue region is the so-called variable region, which interacts with the target epitope. The dark blue region is conserved, it's the same as long as the immunoglobulin is produced by the same species. Normally this region is targeted by secondary antibodies to visualize the binding of the primary antibody.
What are the special challenges regarding the immunochemical investigation of glutamate receptors? Well, first of all, they expressed a very low-level compared to other proteins in the brain. They show a very high-level of sequence homology, so it's virtually impossible to separate them using classical biochemical, biophysical techniques. There are relatively few unique and highly-antigenic regions, which limits our options when it comes to the production of immunochemical tools. As I highlighted in my introduction, they have a very complex structure multi subunit, they can assemble in various ways and they have a very complex molecular organization. Various assembly intermediates exist, for example, it's very difficult to study cell surface expressing functional receptors in isolation, when you also have a large pool of intracellular partially assembled receptors trapped in endoplasmic reticulum. It's very difficult to design studies which will target just one or the other population. Also, most of these receptors are targeted to the postsynaptic entity where they interact with coupling proteins, and linked to the cytoskeleton, which means they are purely accessible for these relatively large antibodies.
There are also several covalent modification sites in the glutamate receptors, for example, about a half a dozen glial conservation sites exist in each inotropic glutamate receptor subunit. In the intracellular domain there are several phosphorylation sites, which can interfere with antibody binding, and there are also protein-protein interactions which can interfere with the binding of antibodies. You need to be aware of the inter-species differences, so you need to look at carefully the epitope region and if an antibody hasn't been tested in a particular species, you need to first confirm that the epitope region is conserved. Then like any other membrane protein, there are issues regarding the presentation of the epitope, so these receptors are present differently if they are in the solubilized or denatured form, or after fixation for immunohistochemistry.
So the first decision is whether you want a polyclonal antibody or a monoclonal antibody when it comes to selecting one from a company. So what are the advantages for using a polyclonal antibody? Usually, it's relatively straightforward to produce these, and these antibodies interact with several different binding sites of the same antigen. Usually, it's an advantage that they are available from different host species. The reason is why it's an advantage, because that can facilitate multiple labelling of the same samples, you can perform co-localization studies or simultaneous studies of two samples using immunofluorescence, for example. Generally, the antibodies are available from rabbit, goat, guinea pigs and various combinations of these can be used for multiple labelling.
In terms of limitations, due to the multiple epitopes it's very difficult to pin it down exactly where the antibody binds. Also, there is a limited supply and you can easily run out of the immunoserum if you use for large scale experiments. The problem is that it's difficult to replace these antibodies, because there are individual variations in immunoresponse, so even if you apply exactly the same immunization protocol, you could end up with fundamentally different antibodies. That's why when you switch the different lot number or different animal, it's highly recommended to rerun some of the specificity tests before you rely on these reagents.
Monoclonal antibodies, they are highly specific for a single epitope and once you've reached the hybridoma stage in antibody production, you no longer need to immunize animals, so then you can grow these cells in cell cultures and harvest the supernatant, and recover the antibodies from there. That provides a really consistent, theoretically endless supply of antibodies and the reaction with these antibodies tends to be more reproducible.
There are limitations and that's partly due to the nature of how these antibodies are produced. Normally, these are produced in mouse, but Abcam also supplies rabbit monoclonal antibodies, which widens the choice somewhat.
So the next decision after you've decided which one seems better, monoclonal or polyclonal, it's to look into the details how these antibodies will produce, because that has various implications. In an ideal world, it would be nice to have antibodies against the native proteins, and purify the proteins in high purity and use that for immunizations and that would be an ideal scenario. As I mentioned, it's impossible to purify glutamate receptors due to the low expression level and also because of the highly similar nature of these proteins. We need to use a more indirect approach, which is based on either synthetic peptides or fusion proteins.
Let's start with the synthetic peptides, so these are chemically synthesized from amino acids and these are relatively small segments of the amino acid sequence, usually a dozen amino acids. They are relatively small molecules about 1-2 kDA. They are not particularly good immunogens, so they need to be linked covalently to a carrier protein which will increase their size, and be present and it's a more potent immunogen. So the advantage of using these, that you have a fairly good idea where these antibodies are going to bind, because you selected the segment and it provides great predetermined specificity, because usually these antibodies are raised against the unique region.
There are also substantial limitations and mainly the first one is a big problem, because of the small size of these proteins quite often these don't interact very well with the native glutamate receptors. Because of the presence of the carrier proteins, almost always you need to perform affinity-purification before you can use these antibodies, and usually this is done by the company who supplied this, but it's well worth understanding how this process works. Often, you'll need to keep this in mind when you prepare your samples, because you need to present your epitopes in such a way that these antibodies can recognize it. Because these are raised against a short sequence, these antibodies tend to be sequence-specific, so you need to expose this sequence either by denaturing your protein or digesting it to provide better access to the corresponding region.
Fusion proteins, usually they represent a longer stretch of the target protein, something like 25 up to 100 amino acid residues, so the larger antigens are better immunogens and they adapt the conformation better. Normally, they induce a more robust immunoresponse and higher quality antibodies, and these antibodies are normally raised against multiple epitopes along the sequence.
That's also a little bit of a downside, because if your ultimate aim is to have subunit-specific antibodies, this may compromise this due to the large size, so you may end up with an antibody which reacts with related subunit isoforms. Due to the size, you're not going to know exactly where your antibody binds, and because there are domains in this fusion protein like glutathione S-transferase, which can also induce immunoresponse, you need to perform affinity-purification, so you only collect antibodies which are raised against the receptor specific for that segment of the fusion protein.
I mentioned purification, so what are the commonly used procedures? The first one is fairly straightforward, and that leads to the precipitation of all immunoglobulins from the immunoserum, and, for this, usually companies use saturated ammonium sulphate. The same concept applies to affinity chromatography using protein A and protein G columns that recover all immunoglobulins, not just those which are selected for glutamate receptors. If you want to recover those which were only raised against your target antigen, then you need to perform immunoaffinity chromatography where you immobilize the antigen that pass through the immunoserum, through this column with the specific antibodies and it will be retained by the column and then you can wash it and elute the specific antibodies using low pH lysine buffer, for example. If you, after the purification, still have substantial background labelling, it's usually a good idea to pre-adsorb the antibodies. For this you can use protein extract from different samples where glutamate receptors are not expressed, for example, liver tissue.
So how do you validate your antibodies? This has to be done for your specific application. Usually, the first step is to test whether you get the decent signal response out of this immune titer and that's done by an ELISA assay. But that's not going to provide information about the specificity of the antibody, this will only indicate the strength of the immunoresponse. A lot more informative are immunoblots where you have a membrane fraction and then you label it with the antibody, and the antibody should identify a single band at the correct molecular weight, which will provide a good indication of antibody specificity. We find that in our laboratory we frequently use histoblots, and these are essentially proteins transferred from unfixed cryostat sections onto a nitrocellulose membrane, and stain pretty much exactly the same way if you stain immunoblots. That will give an overall pattern of the distribution of your target epitope, and that could be very useful, particularly if in the literature you can find alternative techniques like autoradiography in situ hybridization, and put it next to your histoblots that will help to validate your staining. Immunoprecipitation can be used to identify interaction partners for various antibodies, and like histoblots, conventional immunocytochemistry/immunohistochemistry can provide information about overall distribution of immunoreactivity obtained with your antibody.
Controls; these are very important to validate your antibodies. First, you need to start with a cell which is known to express your target protein, and for this you can use transfected cells, but most glutamate receptors express well in HEK293 cells, even embryonic kidney cells. You can use non-transfected cells as negative controls. A very useful control to use brain tissue obtained from transgenic animals where only the target protein is knocked out, and you compare it to wild-type and all the labelling should disappear when you immunolabel these transgenic brain samples. But using common sense can help you to validate an antibody, for example, the immunoreactivity you've obtained from dissected brain regions, or on histoblots should correspond well to regional development variations as published by other techniques like in situ hybridization or autoradiography. If you can, it's useful to obtain two different antibodies against the same target, and that will help to validate the reaction pattern you obtain. It's frequently used, but not a foolproof specificity test, is to block the antibody antigen interaction, because this will only address the issue whether your antibody reacts with a particular antigen, so it's not the ultimate proof of antibody specificity. But, still, it will provide information about the method specificity of your protocol, so you can use the antigen synthetic peptide or fusion protein to block antibody binding to your target tissue. Similar, along the same lines, to remove the primary antibody or denature it, for example, at 60°C at five minutes will do the trick to inactivate antibodies, and that will prove whether you're washing that secondary antibodies provide any significant background labelling, so for troubleshooting this can be very useful. For polyclonal antibodies, it's highly recommended to hang on to some of the pre-immune serum, which was obtained from the animal before the immunization protocol started, because later this can be used to validate your immunolabelling. Other fairly solid proof, if you can pull out the target protein and verify it using mass spectroscopy.
Here is just an example from our lab where we tested the two antibodies against different kainate receptor subunits GluK1 and GluK2. So, first, we tested them against the rat brain and brain fraction, where both identified a nice single band at the right molecular weight region. When we pre-adsorbed these antibodies using the corresponding fusion protein which was used to raise these antibodies, all labelling disappeared. Then next we wanted to establish the subunit selectivity of these antibodies, so we transfected human embryonic kidney cells with individual subunits, and we probed them in parallel with the same antibody. As predicted, because these antibodies were raised against the unique region, they only interacted with the corresponding subunit. We performed additional control experiments using wild-type and knockout brains, and you can see here it's very reassuring that all labelling disappeared when we probed knockout brain samples.
Here at the bottom you can see some histoblots and these are the newly-tested antibodies, and you can see here that the pattern of immunolabelling resembles the pattern that we obtained with the previously well-characterized antibodies raised against the related subunits.
So what are the common problems and pitfalls? The main problem, and that's particularly valid for anti-peptide antibodies, is the lack of cross-reactivity with the native glutamate receptor proteins. Then, quite often, if you use fusion proteins you lose the subunit isoform specificity. Often you can see non-specific cross-reactions with unrelated proteins, and it can be difficult to get rid of. For all polyclonal antibodies you need to be very aware of batch-to-batch variations, and if you switch them you supply a new batch and you need to revalidate your antibody. There are also species differences, and that can apply as much to the epitope region, but also to the antibody raised against the same epitope may behave differently if it was developed in a different species. Sometimes you end up with high background staining where probably reabsorption of the antibody could help.
So now we have a few special issues with the particular applications of anti-glutamate receptor antibodies. Quite often we use these antibodies for immunoprecipitation, and this is very useful when you want to study protein-protein interactions, or you want to understand post-translational modification of these receptors like phosphorylation, dephosphorylation, glycosylation, ubiquitination, SUMOylation or enzymes. So here it's really useful if you can apply two different antibodies, preferably developed in two different species. One for the immunoprecipitation step and another one for the identification of the immunoprecipitated sample. The advantage of this is because if you would use the same antibody for both, immunoprecipitation and labelling, usually you get the really nasty, non-specific band due to the presence of the IgG molecules. You need to be aware of the incomplete solubilization of glutamate receptors. Quite often in the literature you can see papers where it's obvious that glutamate receptors are not fully solubilized for immunoprecipitation. What I mean by this, that before you start immunoprecipitation you need to make sure that all the insolubilized receptors are fully removed. That means you need to spin them at high speed above 100,000g in an ultracentrifuge, and only after that you can start doing immunoprecipitation, otherwise, you might end up with artefacts. It's often overlooked, but well-documented in the literature, the differential solubility of unassembled and assembled subunits of various receptors, and that's particularly relevant for NMDA receptors. Other glutamate receptors tend to aggregate quite easily, for example, group two metabotropic glutamate receptors are usually quite difficult to keep in the monomeric form. If you are planning to compare different immunoprecipitated samples, you need to make sure you use enough antibodies to have a really complete immunoprecipitation of glutamate receptors.
Quite often we use covalent modification of cell-surface-expressed proteins to get information about receptor targeting. For this cell-surface biotinylation, or surface of crosslinking is used routinely. These reagents all interact with amino acid side chains, so if, for example, your antibodies raised against extracellular domain and contains lysine, arginine, asparagine, glutamine, that may alter the antibody binding to the receptors. So for these applications it's much better if you use antibody raised against the epitope which does not contain these amino acids, and these are more preferable to use antibodies against intracellular C-terminal epitopes.
Similar would apply to chemical fixation with a formaldehyde, glutaraldehyde for immunocytochemistry, immunohistochemistry to preserve the structure of your tissue sample. You need to fixate and, unfortunately, has introduced covalent modifications can alter the antibody binding site, and it can prevent the antibody binding. Also, if you are planning to use an intracellular domain-specific antibody, then you need to make sure that after fixation you permeabilize the membrane, otherwise the antibody is not going to have access to these domains.
Also, if you use the extracellular domain-specific antibody, fixation can alter the structure of protein and quite often you need to rely on epitope retrieval to loosen up the structure, and expose the regions or antibody reaction, and this can be done by microwave treatment or pepsin digestion.
Then, more recently, proteomics are used in combination with antibody precipitation of different receptor complexes to analyze the native receptor. Here, the common problem is a very low yield, so you need to scale it up considerably to get the meaningful amount of sample. Also, you need to try to find an antibody which reacts well away from the interaction sites, so, for example, for these experiments extracellular and domain-specific antibody would be more desirable because that's not going to interfere with interactions of the C-terminal domain.
Finally, just to conclude, at present there is no universal validation for antibodies. If an antibody works in one system, for example, it gives a really nice, clean band on an immunoblot and it doesn't mean it will work equally well, equally specifically for immunolocalization studies. So you need to validate it for your particular application, so that is a method specificity for each antibody. You need to get a lot of information about the antibody, and these are available from the company who supplied this, like antibody binding site, sequence of that region. Based on this information you can make an informed decision whether these antibodies are likely to work in your system. Also, you need to be aware of the antibodies are just one side of the story, and how you present your immunogen is another. If you have a covalent modification you need to be aware of these issues, and select the appropriate antibody for your application.
Without appropriate validation, unfortunately, artefacts can slip in and that can be really misleading, so you need to make sure that you validate your antibodies for your specific application. But if you follow these rules, then these immunoreagents are extremely useful and can lead to significant discoveries. So thank you very much and now I'll hand it over to Elizabeth who is going to talk a little bit about the products supplied by Abcam.
ED: Thank you Elek for such an interesting talk. I would like to take this opportunity to tell you a little about the resources that we have here available at Abcam to aid your glutamate research. Abcam offers a diverse range of primary and secondary antibodies, small molecule agonists and antagonists, ELISA kits, proteins, peptides and many more products. Throughout the next few slides I will give you more details about our products, and I'd like to encourage you to take a look at the website to see information on the full range available. Abcam offers an extensive range of both primary and secondary antibodies, which are suitable for a variety of applications. As Elek mentioned earlier, rabbit monoclonal antibodies offer a number of advantages over traditional mouse monoclonal products. Abcam's range of rabbit monoclonal antibodies provide better antigen recognition than the polyclonal counterparts. The rabbit immune system generates greater antibody diversity and optimizes affinity by the mechanisms that are more efficient than those of mouse and other rodents. This increases the possibility of obtaining a functional antibody that will work in a variety of applications.
Alternatively, if you need to develop an antibody for a hard to characterize target, or are looking to develop phospho-antibodies or other modifications, you may want to use our custom rabbit monoclonal service. Our custom antibodies development process is flexible and tailored to your specific needs. The services offered include: rabbit monoclonal and polyclonal antibody generation, immunogen design and preparation, immunoassay development, IgG cDNA and cloning recombinant proteins. Our PhD level project managers are very responsive in providing updates throughout the project, and this issue with any technical details. Our custom antibody team has demonstrated a record of success helping customers develop rabbit monoclonal antibodies. More than 550 institutions and 150 pharma and biotech companies have benefited from our custom antibodies. A list of citations referencing custom rabbit monoclonal antibodies, as well as our growing list of customer testimonials can be found on our webpage.
Abcam offers more than 100 antibodies for inotropic glutamate receptors. Our range covers AMPA, kainate, as well as NMDA receptors, and their subtypes. All our antibodies are tested in multiple applications and species. We also offer antibodies raised in different hosts, thus affording greater flexibility for the design of multicolor IHC experiments. Examples of our inotropic glutamate receptor antibodies can be seen here, and from left to right the first is an example of an AMPA receptor in rat cerebellum sections. The image in the middle is an example of a mouse monoclonal antibody to the kainate receptor in rat cerebellum sections. The image on the right is an example of a rabbit monoclonal to the kainate receptor in human colon sections. All of these antibodies will react with human, rat and mouse.
We also offer the same variety in the metabotropic glutamate receptors. The image on the left is an example of rabbit polyclonal to mGluR4 in mouse cerebellum. This antibody can be used in human, rat, cat and chicken. The image in the middle is an example of a chicken polyclonal to mGluR5 in adult mouse cochlear ganglion. The image on the right is an example of a guinea pig polyclonal to mGluR6 in the mouse retina. In addition to our range of antibodies, Abcam offers a range of pharmacological tools and these include over 100 glutamate receptor agonists and antagonists to all glutamate receptor subtypes, as well as enzyme inhibitors and ion channel modulators. These products are available both selective for specific glutamate receptors, as well as non-selective ligands for targeting groups of receptors. We also have a number of negative and positive allosteric modulators. These tools are appropriate for both in vitro and in vivo applications, including orally active and blood-brain barrier permeable products.
A selection of our standard and novel tools can be seen here. We have a range of agonists and antagonists specific for NMDA, AMPA, kainate and MGlu receptors. Some of our popular compounds include D-AP5, a competitive NMDA glutamate site antagonist, and NBQX disodium salt are water-soluble, potent, selective and competitive AMPA and kainate receptor antagonists. For information on purity, solubility and for references and citations, please view the datasheets on our website. These compounds have been cited in a number of papers investigating behavior, function, localization and expression studies in journals such as Nature Neuroscience.
Agonists and antagonists have contributed hugely to this discovery and understanding of glutamate receptor subtypes. In more recent days, these compounds have an increasing importance in drug discovery and therapeutics. However, there are a number of considerations to remember when choosing the right agonist and antagonist for your research. For example, is the product selected for your receptor or receptors of interest? Some research may require or tolerate a less selective agonist that targets a number of isoforms, whereas in other cases it may be essential for a highly selective and subtype-specific product. The potency that can be used is affected by cell type species and the type of experiment, for example, with in vitro versus in vivo research. With in vivo research the root of administration must also be considered, for example, if the product active is following oral administration and does it cross the blood-brain barrier, it may also be metabolized by the body before it reaches the target site. All of our products we always recommend that you perform a dose response or DMPK study, to ensure that your product is being administered at the best concentration for your system.
If you are limited on time, we now also offer a range of glutamate agonists and antagonists made up to give an exact ready-to-use concentration upon the addition of 1 ml of water. This means you will not have to worry about complicated molar calculations and can spend more time on your experiments. These products are available in a range of concentrations, including 100 mM and 50 mM. Please visit the website for further details, and to view the full range.
As well as our products you may like to take advantage of our extensive resources, all available from the website. We have a wide range of posters and pathway cards covering topics such as neuroscience, epigenetics and cancer. Protocol books are also available, including western blotting and immunohistochemistry and contain methods and troubleshooting tips. All of our product datasheets contain relevant up-to-date information to assist you in selecting the right product, including specific references, applications and species. If you have used one of our products before and would like to share experience with other users, we encourage you to submit an AbReview which are easy to complete and will earn you money off your next purchase. All of our receptor agonists and antagonists come at exceptional purity determined by HPRC, NMR and mass spectrometry, as well as other techniques.
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After today's webinar we will be emailing all participants a PDF copy of the presentation, and a video will be uploaded to our event webpage. All of our past webinars can be viewed online, including a number of videos covering applications and techniques, as well as other research hot topics like obesity and post-translational modifications. Once again, I would like to thank you all for attending, and I would now like to pass you back over to Elek who will try to answer as many of your questions with our remaining time.
EM: Thank you, Elizabeth. We've received a few questions, the first one is from Lucas: Can I distinguish synaptic versus extrasynaptic NMDA receptors using immunohistochemistry? Usually for this level of analysis you need high resolution, so I would recommend to use electron microscopy. For electron microscopy pre-, post-embedding immunogold techniques I use routinely for identification of synaptic versus extrasynaptic. Sensitivity of these techniques varies. If you are looking for a high sensitivity technique, peroxide-based localization techniques give a good overall indication, but you should remember that the reaction and product can diffuse away from the actual antibody binding site, which can compromise the resolution of your technique. If you use synaptic markers it's not absolute, because this is, again, pushing the resolution limit of light microscopy. But with confocal microscopy in combination with synaptic markers, again you can assess synaptic versus extrasynaptic NMDA receptors.
The next question is: Can you explain what histoblots are and why do you use this technique for the validation of antibodies? As I mentioned, these are relatively straightforward techniques to identify regional distribution, so it's not going to provide high-resolution images, but it's good enough to compare the distribution of the immunostaining previously established better and obtained with, for example, autoradiography or in situ hybridization. So it is basically an in situ blotting technique, so we cut ten micron on fixed cryostat sections, we press it against the nitrocellulose membranes. Some proteins will be transferred onto these nitrocellulose, and then from that point on we treat this pretty much the same way as you would treat the immunoblots, so you would block it in milk powder containing buffer and then at the primary, secondary antibody and so on. Probably the only difference is it's tried ECL-based techniques, but that doesn't give good enough resolution, so it's better to stick with good old-fashioned alkaline phosphatase-based reactions. So we use alkaline phosphatase-conjugated secondary antibodies, and those provide a fairly nice image that you can scan in, analyze. We find that you can even compare different brain samples as long as you cut sections exactly the same way.
The next question is: Can you recommend a good epitope retrieval technique for glutamate receptors? Personally, I have relatively limited experience with these, but from the literature I can recommend short pepsin digestion, this produced a pretty impressive immunostaining of pepsin digested samples.
The other question is: How can you prevent degradation of metabotropic glutamate receptors on immunoblots? Well, I agree that's a tricky problem, some MGluRs, for example, mGluR3, mGluR1 tend to aggregate quite a lot, so it's very difficult to identify them in monomer form. I think the trick is not to heat them up when you load them onto gel, and when you prepare your membrane samples for immunoblotting, then use protease inhibitors in all membrane and lysis buffer. If I remember correctly, don't use β-mercaptoethanol you need to use dithiothreitol (DTT) instead for gel separation.
There is a question: Which glutamate receptors are known to be present in glial cells? Well, it largely depends on the developmental stage, because immature oligodendrocytes, for example, will express a whole range of neurotransmitter receptors, including calcium permeable AMPA receptors, kainate receptors. They also express some of the metabotropic glutamate receptors like mGluR3, mGluR5, but the expression level of these receptors tend to downregulate as these progenitors mature, differentiate, but immunohistochemical studies identified these receptors even in fully matured central nervous systems. I think we've ran out of time now. Thank you very much.
Thank you, Elek and Elizabeth for your presentations today. Unfortunately, due to time restrictions we have not been able to answer all the questions we've received. For those whose questions were not answered, we will come back to you shortly via email with a response to your question. As Elizabeth mentioned, after this webinar a PDF copy of the slides will be emailed to you. If you have any further questions about what has been discussed at today's webinar, or have any technical queries, please do not hesitate to contact our scientific support team who would be very happy to help you. They can be contacted at firstname.lastname@example.org. We hope you have found this webinar informative and useful to your work, and we hope we can welcome you to another webinar in the near future. Thank you, again, for attending and good luck with your research!