Videos uploaded by user “Cell Signaling Technology, Inc.”
Epigenetics Overview
Brief introduction to epigenetic regulation including the the two states of chromatin: euchromatin and heterochromatin. Transcript Epigenetic regulation encompasses a number of different modifications to chromatin. These include methylation of the DNA on cytosine bases, a modification that can further be oxidized, as well as modification of the histone tails that emanate from the core of the nucleosome. The tails of core histones labeled here can be altered with distinct chemical modifications including methylation of Histone H3, acetylation of Histone H4, and phosphorylation of Histone H2B. Euchromatin is often characterized by a more open and accessible state of the DNA one in which transcription factors have access to their cognate binding sites and can therefore recruit enzymes like histone acetyl transferases that acetylate histone tails and activate genes by recruiting components of the basal transcriptional machinery, including RNA polymerase. Heterochromatin ,in contrast, is thought to be characterized by a more repressive tight bundling of nucleosomes which impedes transcription factors from gaining access to regulatory sites on the DNA. Methylation of cytosine bases and regions called CpG Islands is a hallmark of transcriptionally repressed heterochromatin. These methylated cytosines in turn recruit proteins like MeCP2 (Methyl CpG binding protein 2) and HP1 (Heterochromatin Protein 1). These proteins are thought to maintain a repressive state of chromatin by inducing histone deacetylation by HDACs as well as histone tail methylation by histone methyltransferase enzymes.
Flow Cytometry Animation
Brief introduction to flow cytometry from Cell Signaling Technology (CST). Visit 👉https://www.cellsignal.com/flow for more information on validated products and optimized protocols for flow cytometry. Transcript: Cell processes are controlled by complex and dynamic intracellular signaling networks. Surface receptors pass information into the cell by modifying cytoplasmic proteins. These modifications include phosphorylation,. methylation, acetylation, which control a broad range of cellular functions, including growth, development, and apoptosis. Disruption of signaling networks often leads to disease. To understand signaling processes and normal and pathological conditions, identification and quantification of signaling proteins is necessary. Flow cytometry is ideal for such a task. Flow cytometry is a laser based technology widely used to phenotype cells using antibodies directed against surface markers. It can also allow simultaneous analysis of multiple intracellular targets using antibodies that detects and signaling proteins, their post-translational modifications, epigenetic modifications, and downstream transcription factors. The first step of intracellular flow cytometry is fixation of the cells using formaldehyde, which immobilizes proteins and preserves transient signaling events. Cells are then permeabilized with methanol or detergent, allowing antibodies to access to the intracellular space. Optimal fixation and permeabilization conditions depend on the location and biochemistry of the target epitopes. It should be carefully considered to ensure successful antigen detection, especially when staining surface- and intracellular proteins simultaneously. Since multiple signaling proteins and surface antigens can be identified using fluorescently distinct antibodies, flow cytometry can readily characterize signaling profiles at the single-cell level within phenotypically distinct populations. This allows the characterization of the relationship between intracellular targets and enables detection of signaling events from rare-cell populations within a heterogeneous sample. Multiplex analysis of these protein targets saves time and resources while producing data-rich results, making flow cytometry an invaluable tool to unravel the complexities of cell signaling.
Apoptosome Formation
A brief introduction to Caspase-mediated formation and activation of the apoptosome. Cytochrome c released from the mitochondria during apoptosis leads to amplification of the caspase signaling cascade via formation of the apoptosome. A radially-symmetric structure, the apoptosome assembles upon binding of cytochrome c (blue) to Apaf-1 (beige), followed by recruitment of caspase-9 (red). Once assembled, the apoptosome's activated caspase-9 cleaves and activates cytosolic caspase-3 (red, floating) and rapidly amplifies the caspase cascade. This leads to the destruction of numerous intracellular targets by caspase-3 including the actin cytoskeleton (green).
Immunohistochemistry Protocol for Paraffin embedded Tissue Sections
IHC Protocol Video for Paraffin-embedded Tissue Sections from Cell Signaling Technology (CST) 👉 CST Protocols: http://cellsignal.com/protocols 👉 Download the Guide to Successful IHC: https://learn.cellsignal.com/cst-application-guides 👉 Technical support: http://cellsignal.com/support Immunohistochemistry (IHC) is a powerful microscope-based technique that uses an antibody to view a specific protein in biological tissue. It is often used to diagnose abnormal cells in solid tumors, visualize molecular markers for cellular events like apoptosis, and monitor the localizations and expression of biomarkers. However, individuals new to this method can struggle due to the many steps in the procedure that can each add variations that will impact staining. Cell Signaling Technology (CST) scientists have determined optimal conditions for CST antibodies developed and validated in-house for IHC. In this video, we demonstrate the IHC protocol we have optimized for formalin-fixed paraffin embedded (FFPE) tissue sections so you can replicate results in your laboratory and obtain consistent results. We will first describe how to prepare your sample and deparaffinization/rehydrate the FFPE samples followed by the antigen unmasking step, the most difficult step in an IHC protocol. Next, we will review chromogenic staining with the SignalStain DAB Substrate Kit followed by how to mount sections to a coverslip before viewing on a microscope. Finally, we describe how using different conditions than those recommended each antibody Product Data Sheet, like changing the recommended antibody diluent, can affect your final results. If you’re interested alternate protocols including performing IHC on frozen tissue samples visit the Protocol at the Cell Signaling Technology website (http://cellsignal.com/protocols).
PI3K / Akt Upstream Signaling from Cell Signaling Technology, Inc.
The PI3K/Akt signaling pathway controls a variety of cellular functions including glucose metabolism, cell survival, proliferation, and invasiveness. Due to its role in these important processes, proper regulation is critical as constitutive activation of Akt has been observed in a variety of cancers including neuroblastoma, breast cancer, and prostate cancer. In this video, we use pan and phospho specific Akt antibodies from Cell Signaling Technology (CST) for an in-depth review the upstream signaling events that regulate Akt using confocal immunofluorescent analysis. The PI3K/Akt pathway is initiated when growth factors or cytokines signal through membrane receptors including receptor tyrosine kinases (RTKs), cytokine receptors, G-protein coupled receptors, B-cell receptors, and integrin receptors. These receptors signal through PI3K which in turn phosphorylates PIP2, converting it into PIP3. PIP3 then binds to the PH domain of Akt, anchoring it to the cell membrane where the kinase PDK1 and mTORC2 complex activates the membrane bound Akt through phosphorylation on Thr308 and Ser473 respectively. This triggers activation of downstream proteins (substrates). PI3K/Akt signaling is inactivated when the phosphatase PTEN converts PIP3 back to PIP2, leaving Akt in its inactive dephosphorylated state. Visit http://www.cellsignal.com to see antibodies for the study of Akt Signaling.
Pluripotency at the Molecular Level from Cell Signaling Technology, Inc.
Brief introduction to the predominant signaling pathways in human embryonic stem cell (hESC) pluripotency: TGF-β, FGF and Wnt. Pluripotency at the molecular level. At the molecular level, the predominant signaling pathways involved in human ESC pluripotency and self-renewal, are TGF-β, which signals through SMAD2 and 3 and FGFR, which activates the MAPK and Akt pathways. The Wnt pathway also promotes pluripotency through activation of β-catenin. Signaling through these pathways results in the expression and activation of three key transcription factors Oct-4, Sox2 and Nanog. These transcription factors activate gene expression of ESC-specific genes, regulate their own expression and also serve as hESC markers. Other markers use to identify human ESCs are SSEA3 and 4 and TRA-1-60 and TRA-1-81. Pluripotency and self renewal are inhibited by the BNP4 pathway which uses SMAD1, 5 and 8 to block transcription factor expression. The pathway shown is for human cells, pluripotency in mice involved slightly different upstream signalling primarily the LIF leukemia inhibitory factor and STAT3 pathway, but results in expression of the same key transcription factors. Confocal immunofluorescent analysis showing expression and nuclear localization of the transcription factors Oct-4, Sox2 and Nanog in NTERA2 cells. Oct-4, Sox2 and Nanog antibodies have been labeled green. Actin filaments have been labeled red with Dy-554 Phalloidin. NTERA2 to is a human embryonic carcinoma cell line that expresses the same markers as ESCs. Note the presence of green signal in these cells. Hela cells are a non-pluripotent epithelial cell line used as a negative control and do not express these stem cell markers.
Western Blotting Protocol
In this video we take you through all the steps in our Western Blot (WB) Protocol so you can replicate our procedure and get reproducible and reliable results. 👉 CST Protocols and Troubleshooting: https://www.cellsignal.com/protocols 👉 Western Blot Troubleshooting Video: https://www.youtube.com/watch?v=CQ9gGb8bqn8 👉 Subscribe: https://youtube.com/user/cellsignaldotcom?sub_confirmation=1 Western blotting is a widely used immunoassay used by research scientists to monitor expression of a specific protein in a complex cell or tissue extract. It utilizes antibodies that recognize a specific protein of interest or a post-translational modification like phosphorylation, acetylation, methylation, and ubiquitination. At Cell Signaling Technology (CST), we perform thousands of Western blots daily using a protocol that has been optimized for over a decade to develop and validate antibodies with exceptional specificity, sensitivity, and reproducibility. We first list all the the solutions and reagents you’ll need. Then, we give detailed descriptions on how to prepare the sample, perform the protein blot and immunoassay, and detect proteins chemiluminescently. Finally, we describe critical experimental steps in Western blotting and explain how small changes to the protocol, like changing incubation times or dilution buffers, can affect the final outcome of your blot. To learn more about how to generate publication ready Western blot data, check out our troubleshooting video (https://www.youtube.com/watch?v=CQ9gGb8bqn8). If you’re interested in alternate protocols, including preparing your sample with immunoprecipitation, re-probing your membrane, and detecting your protein using fluorescence detection, visit the Protocol at the Cell Signaling Technology website (https://www.cellsignal.com/protocols).
Western Blot Troubleshooting Guide
This video guide provides tips to help you diagnose problems and solutions to ensure reliable Western Blotting (WB) results. 👉 Download the Guide to Successful Western Blotting https://learn.cellsignal.com/western-blotting-white-paper-request-web-referral 👉 CST Protocols and Troubleshooting: https://www.cellsignal.com/protocols 👉 Western Blot Protocol Video: https://www.youtube.com/watch?v=yUstng0npaY 👉 Subscribe: https://youtube.com/user/cellsignaldotcom?sub_confirmation=1
David Sabatini, Ph D , M D , Growth By The mTOR Pathway
David Sabatini, Ph.D., M.D. of the Whitehead Institute at MIT discussed his work defining the role of mTOR in the amino acid sensing pathway.
What is a Stem Cell? from Cell Signaling Technology, Inc.
Embryonic stem cells, also called ESCs or ES cells, are cells derived from the inner cell mass of a blastocyst. Embryonic stem cells have two unique features. They are pluripotent and have the ability to self-renew. This video describes what an embryonic stem cell is, why these cells are unique, and why they are important for basic and clinical research. Visit http://www.cellsignal.com to see antibodies directed against stem cell and lineage markers.
The Study of Stem Cells - Part 1 of 3
Section One - This learning video from Cell Signaling Technology, Inc. (CST) provides an overview of embryonic stem cell markers (ESCs) and ESC markers. This section also includes information regarding induced pluripotent stem cells, also referred to as iPS cells, and CST's new StemLite™ Pluripotency Kit #9656. Visit http://www.cellsignal.com to see antibodies directed against stem cell and lineage markers.
The Study of Stem Cells - Part 2 of 3
Section Two - This learning video from Cell Signaling Technology, Inc. (CST) covers information concerning Stem Cell Differentiation along specific lineage pathways and corresponding specific lineage markers. Visit http://www.cellsignal.com to see antibodies directed against stem cell and lineage markers.
Formaldehyde vs. alcohol fixation for immunofluorescence (IF) | CST Tech Tips
Let's explore the difference between crosslinking, aldehyde-based fixation and alcohol-based fixation for immunofluorescence (IF). 👉Subscribe: http://youtube.com/user/cellsignaldotcom?sub_confirmation=1 👉CST Protocols and Troubleshooting: http://cellsignal.com/protocols 👉Get in touch with a CST scientist: http://cellsignal.com/support Transcript: What's the difference between crosslinking aldehyde fixatives and alcohol based fixatives for immunofluorescence? Hi, I'm John Burford, senior research associate on the Immunofluorescence Validation team here at Cell Signaling Technology, and this is CST Tech Tips. Fixation is an important step in the IF protocol. Fixation works by stopping the activity of endogenous proteases in the sample so that proteins aren't lost, and by preserving the sample architecture so you can then introduce antibodies to detect your protein of interest. Ideally, fixation would not alter the ability of antibodies to detect proteins, but unfortunately fixatives do have an impact on the epitopes, and can also affect the fluorescent properties of the sample itself. Because of this, it's important for you as an antibody user to understand the different types of fixation approaches. For IF, there are two main classes of fixatives. First, are aldehyde based crosslinking fixatives which form chemical bridges between the lysine groups of proteins, providing structural stabilization to the cell. There are three potential challenges with crosslinking fixatives. One, when proteins are packed tightly together and highly crosslinked, it may be difficult for the antibody to access the protein of interest. Second, if the antibody does make it to the protein, the epitope it recognizes may be chemically altered, effectively inhibiting antibody binding. Third, aldehyde fixation can create autofluorescent byproducts in the sample that increase nonspecific background in IF. This can be somewhat avoided by choosing longer wavelength, lower energy fluorophores. The second type of fixation approach is alcohol based organic solvent fixation. This works by stripping the proteins of their hydration barrier, effectively dehydrating the sample. This washes away soluble proteins, but precipitates the proteins that remain and alters their tertiary structure. This can help antibodies access otherwise hidden epitopes, at the cost of losing soluble proteins. Here at CST, we test all IF antibodies so you can be confident they'll work with the recommended fixation protocol. When designing your IF experiments, check the product pages at http://cellsignal.com for the appropriate protocol for your antibody. Any time you have questions about an antibody or a protocol, you can reach out to one of our scientists, such as myself, at http://cellsignal.com/support. For more tech tips videos, subscribe to our channel, and we'll see you next time. Good luck with your experiments, thank you. About CST: Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high-quality research tools to the biomedical research community. Our employees operate worldwide from our U.S. headquarters in Massachusetts, and our offices in the Netherlands, China, and Japan. http://cellsignal.com/about
About Cell Signaling Technology (CST)
Founded by research scientists in 1999, Cell Signaling Technology (CST) is a private, family-owned company headquartered in Danvers, Massachusetts with over 400 employees worldwide. Active in the field of applied systems biology research, particularly as it relates to cancer, CST understands the importance of using antibodies with high levels of specificity and lot-to-lot consistency. It’s why we produce all of our antibodies in house, and perform painstaking validations for multiple applications. And the same CST scientists who produce our antibodies also provide technical support for customers, helping them design experiments, troubleshoot, and achieve reliable results. We do this because that’s what we’d want if we were in the lab. Because, actually, we are. See more at: www.cellsignal.com
Tumor Cell Signaling and Metabolism
John Blenis, Ph.D., from Harvard Medical School discussesTumor Cell Signaling and Metabolism
The Study of Akt - Section 2 of 3.mp4
This section of "The Study of Akt" focuses on three different target groups: • Effects on Proliferation and Survival • Effects on Protein Synthesis through Regulation of mTOR • Regulation of Autophagy through mTOR Visit http://www.cellsignal.com to download the Akt Signaling Pathway and to see antibodies directed against Akt.
Robert Weinberg, Ph D , Signals Triggering the EMT and Cancer Stem Cell Formation
Robert Weinberg, Ph.D., Whitehead Institute and Koch Institute at MIT
The Study of Akt - Section 1 of 3.mp4
This section of "The Study of Akt" covers background information on Akt (PKB) as well as upstream signaling. Visit http://www.cellsignal.com to download the Akt Signaling Pathway and to see antibodies directed against Akt.
Which Chromatin Fragmentation Method is Better for ChIP?
While effective, sonication is difficult to control and requires exposing the chromatin to harsh, denaturing conditions (i.e., high heat and detergent) that can damage both antibody epitopes and the genomic DNA. Enzymatic digestion, on the other hand, uses a micrococcal nuclease to gently fragment the chromatin into uniform pieces. Chromatin prepared using this method consistently produces a stronger, more reliable signal, which is especially important if you're investigating low abundance, low stability events like the interaction between a polycomb group protein and a specific gene (e.g., Ezh2 [D] or SUZ12 [E]).
Will drugs that target MyD88 dependent signaling network be useful
Sir Philip Cohen, Ph.D., Sir Philip Cohen, Ph.D., University of Dundee. Will drugs that target components of the MyD88-dependent signaling network be useful for the treatment of Diffuse Large B cell Lympoma?
Milk or BSA? Choosing a blocking protein for Western Blotting (WB) | CST Tech Tips
How does choice of blocking protein - nonfat dry milk vs bovine serum albumen (BSA) - affect your Western Blot (WB) results? 👉Subscribe: http://youtube.com/user/cellsignaldotcom?sub_confirmation=1 👉CST Protocols and Troubleshooting: http://cellsignal.com/protocols 👉Get in touch with a CST scientist: http://cellsignal.com/support Transcript: - What protein should I use to block a transfer membrane for a western blot? My name is Srikanth, I'm a product scientist at Cell Signaling Technology, and this is CST Tech Tips. In regards to western blotting, a common question we always get is, what should I use, milk or BSA to block? So the purpose of blocking step is to reduce the amount of background due to non-specific bonding. Now BSA is only made up of one protein, BSA at 60 kDa, whereas milk is made up of many proteins, all of various sizes. So you get a much better chance to reduce more of the background banding. We recommend that you use 5% milk in TBST, shaken for one hour at room temperature, to block all of our non-conjugate primary antibodies. This includes phospho-specific and total antibodies. Now I can already hear the clicking, comments, and hashtags, asking about "what about the phosphatases in milk?" Well, there are some papers out there that discourage you from using milk for phospho signal. Let me address that by saying that we, in all of our in-house testings, we don't see any of these issues. CST scientists run so many westerns that we end up making milk once, at least once a day, sometimes multiple times a day. Now, if your milk buffer goes unused for a week, two weeks, even longer, that increases your chances of phosphatases affecting your signal. But again, we don't see any of these effects, because we use milk buffer fresh on a daily basis. These are images of product #13038, tested on lysates made from 3T3 cells treated with PDGF. The only difference between the blots is that one membrane is blocked in milk, and the other membrane is blocked in BSA. Clearly, the membrane blocked in BSA has a much higher background. Yet phospho signal is still strong and clean with the membrane blocked in milk. So after the membrane has been blocked, please refer to the product's specific data sheet for the recommended antibody collusion buffer. It's either gonna be milk or BSA, depending on the specific antibody. I hope this has been helpful. For full application specific protocols, they're available on cellsignal.com on specific product page. If you have any other questions, please feel free to contact any of the scientists at CST at https://cellsignal.com/support. For more CST Tech Tip videos, please subscribe to our YouTube page. Good luck with your experiments. And we'll see you next time, thanks. 👉About CST: Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high-quality research tools to the biomedical research community. Our employees operate worldwide from our U.S. headquarters in Massachusetts, and our offices in the Netherlands, China, and Japan. https://cellsignal.com/about
Immunohistochemistry IHC Tips and Techniques
In this video, we give you tips for optimizing your Immunohistochemistry (IHC) techniques based on our experience at Cell Signaling Technology (CST). Immunohistochemistry (IHC) is a powerful microscope-based technique that uses an antibody to view a specific protein in biological tissue. However, there are lots of steps in the procedure that each add variation that can impact final results. Therefore, individuals new to the method can struggle initially. All CST antibodies are developed and validated in-house so if the antibody you’re interested has been validated for IHC for paraffin-embedded samples (IHCP) or frozen samples (IHCF), the product’s Data Sheet will have our optimized assay conditions. However, if you need to deviate from our recommendations for some reason this video gives you tips and suggestions on re-optimizing so you can get reliable, reproducible results. First, we’ll focus on how the different steps of the IHC protocol, like using either citrate or EDTA during the antigen retrieval step, impacts final staining. We’ll also go over ways to validate the specificity of your signal, like using treated paraffin-embedded cell pellets as positive and negative control, so you can trust the accuracy of your results. Finally, we give you examples on how the reagents you use can really make a difference on the strength of your final signal. For more IHC tips, visit www.cellsignal.com http://bit.ly/1JhEn5D
Cell signaling and clues to the origin of the cancer epigenome
2013 Koch Symposium presents Stephen Baylin, M.D., John Hopkins University School of Medicine
The Story of an Antibody
Cell Signaling Technology (CST) follows a stringent antibody development process from conception through clonal expansion, screening, selection and validation. This ensures that the antibody product is highly specific and sensitive, with low cross-reactivity, making it well suited for the intended application. It also ensures it can be consistently produced from lot-to-lot. CST team members certify the product for release, underscoring their personal commitment to the product's quality and performance.
Cellular Signal Transduction Animation: Cytokine Receptor Complexes and Activation
Brief introduction to signal transduction featuring cytokine receptor complexes and activation. This video showcases receptor-ligand interactions resulting in the formation and activation of IL-6Rα/gp130 complexes, which in turn trigger the Jak/Stat signaling cascade and the SHP-2/Erk MAP kinase signaling cascade. NOTE: There is no audio track with this video. Visit http://www.cellsignal.com for more information about Cytokine Receptor Complexes and Activation.
Measuring cellular signaling using MS based proteomics
Steven Gygi, Ph D , Measuring cellular signaling using MS based proteomics
Webinar  Multiplex Immunohistochemistry IHC
This webinar demonstrates how multiplex immunohistochemistry (mIHC) can be used to simultaneously investigate the expression pattern of multiple proteins, involved in immune checkpoint control, within the context of preserved tissue architecture. Key highlights: mIHC allows the simultaneous detection of multiple (more than 6) targets of interest in a species/isotype independent manner while providing ample signal amplification Monoclonal antibodies developed and validated by CST can enable co-detection and spatial characterization of important immune checkpoint control proteins using mIHC.
PhosphoSitePlus® Training Tutorial
PhosphoSitePlus, PhosphoSite for short, is a powerful bioinformatics resource that provides powerful adjunct information for your research related to the structure and function of protein modifications in health and disease. Maintained by scientists at Cell Signaling Technology (CST) with grants from the National Institute of Health, PhosphoSite is continuously updated and has information from over ten thousand literature publications and thousands of previously unpublished mass spectrometry experiments mostly performed at Cell Signaling Technology. In this video, we take you through an overview of the structure and content of PhosphoSite. We’ll then describe how to perform a simple search for a protein or substrates of a specific protein kinase of interest. Finally, we’ll review how to use the powerful advanced search and browse interfaces and review the information you’ll get from a search. At the end of this tutorial, you’ll be able to effectively gain access to all the information available using PhosphoSite.
Advances in AMPK and Autophagy Signaling
The webinar touches upon the following key points: - AMPK is a master regulator of organismal metabolism - Identification and characterization of novel AMPK substrates - ULK1 (aka Atg1) is the critical downstream kinase of AMPK that initiates autophagy - ULK1 inhibitors as potential cancer therapeutics
The Study of Stem Cells
Stem cells are undifferentiated cells that are pluripotent, meaning they can become many different cell types. This pluripotency has caused there to be lots of interest potential stem-cell based therapies for conditions like neurodegenerative disease or diabetes, however controversy over how stem cells are isolated still persists. In this video, Cell Signaling Technology (CST) gives you an overview on the study of stem cells. First, embryonic stem cells (ES cells) and ESC marks will be reviewed along with stem cell pluripotency regulation through expression of key transcription factors like Oct-4 and Sox2. Information about induced pluripotent stem cells (iPS cells) and CSTs StemLight Pluripotency kits shared will also be shared. Next, stem cell differentiation along the three lineage specific pathways, the ectoderm, mesoderm (which differentiate along the mesenchymal and hemangioblast pathway) and endoderm, and corresponding lineage specific markers will be discussed. Finally, and overview on how epigenetic mechanisms like acetylation and methylation remodel chromatin to regulate stem cell pluripotency will be given.
Ask these 3 Questions before your next immunofluorescence (IF) experiment
Before you start your next immunofluorescence (IF) experiment, ask these three questions to ensure you will get reliable results and avoid headaches: 1) Is your antibody specific? 2) Is your antibody supported by an optimized IF protocol? 3) Is your antibody performing consistently? 👉 Learn more: http://cellsignal.com/IFProven 👉 CST Protocols and Troubleshooting: http://cellsignal.com/protocols 👉 Get in touch with a CST scientist: http://cellsignal.com/support 👉 Subscribe: http://youtube.com/user/cellsignaldotcom?sub_confirmation=1
How to perform Antigen Retrieval (AR) in Immunohistochemistry (IHC)  | CST Tech Tips
We'll show you why antigen retrieval (AR) is important for immunohistochemistry (IHC), and give you tips on how to set up Heat Induced Epitope Retreival in your microwave. 👉Subscribe: http://youtube.com/user/cellsignaldotcom?sub_confirmation=1 👉CST Protocols and Troubleshooting: http://cellsignal.com/protocols 👉Get in touch with a CST scientist: http://cellsignal.com/support Transcript: - - What is antigen retrieval? And why is it so important for IHC? I'm Abbey Wells. - And I'm Franny Ambrose. - We are both research associates in the immunohistochemistry group at Cell Signaling Technology. - And this is CST Tech Tips. Antigen retrieval, also known as AR, is one of the factors that can influence the strength of staining obtained in IHC. AR made be accomplished by treating the sample with either heat or enzymes. In this video, we'll focus on heat-induced epitope retrieval, or HIER for short. Antigen retrieval takes place after deparaffinization of the sample and before the addition of antibodies to detect the target proteins. AR removes the cross-links formed as a result of aldehyde-based fixation and unwinds the protein's secondary and tertiary structure, making epitopes accessible to detection by antibodies. This can be particularly important for epitopes buried deep in the protein structure. In your standard HIER protocol, samples are submerged in a buffer and boiled for a period of time, usually 10 to 20 minutes. Heating methods commonly used include a water bath, microwave, or pressure cooker. The level of retrieval can very among these methods, with water baths being the least effective and pressure cooker being the most effective. CST's IHC Protocol recommends using a microwave, which is effective for antigen retrieval, and you probably have one in your lab. A common question we get is what unmasking buffer should I use for antigen retrieval. Many antibodies with work well with antigen retrieval performed with citrate buffer. Often with EDTA, you will observe increased signals, but possibly increased background as well. Antibodies that work with citrate buffer are likely to also work with EDTA, but may require further optimization in order to achieve the best signal-to-noise ratio. Conversely, if an antibody requires EDTA for retrieval, it is unlikely to work with with citrate. The antigen retrival buffer that we recommend with our products can be found on the product's data sheet. Make your 1x citrate or EDTA and fill your container with 200 mL of that buffer. Fill any empty slots in your slide holders with blank slide. This will ensure that the buffer and heat distribution is consistent between experiments. Leave the lid slightly cracked to allow pressure to escape as this ensures the buffer does not boil over and expose the top of your slides. Also, overheating slides can cause a loss of buffer, which may result in tissues drying out. This can yield false negative results. A common misstep in antigen retrival is aggressive boiling. That can cause tissue to detach from the slide. To optimize microwave time and power, we suggest performing a trial run. Use 24 blank slides and 250 mL of buffer and determine how long it takes to reach the initial boil. Next, determine the power level needed to maintain a sub-boiling temperature for 8-10 minutes. Once you have established the conditions for your microwave, perform the retrieval the same way each time, always using the same amount of buffer with empty slots occupied by blank slides. You can find full application-specific protocols for each antibody on its product page at https://cellsignal.com. If you have any questions about an antibody or a protocol, you can get in touch with one of our scientists at https://cellsignal.com/support. And for more tech tips videos subscribe to our channel, and we'll see you next time. Good luck with your experiments! 👉About CST: Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high-quality research tools to the biomedical research community. Our employees operate worldwide from our U.S. headquarters in Massachusetts, and our offices in the Netherlands, China, and Japan. http://cellsignal.com/about
Deciphering the Molecular Pathology of Alzheimer’s Disease
Alzheimer’s disease presents stereotypical pathological features including the abundance of amyloid deposition and tauopathy. Affected brain regions also exhibit reactive astrogliosis, neuroinflammatory responses, and vascular pathology. Neuronal and synaptic loss is also evident. Various mouse lines have been created to investigate Alzheimer’s disease and most of the models exhibit a subset of the defining pathological features of the disease. In this webinar the speaker will discuss the use of Alzheimer’s disease transgenic mouse models to evaluate pathological features by both traditional immunohistochemistry and new tissue processing methods that enable whole- brain imaging. These immunolabeling methods further the investigation of molecular and neural circuit mechanisms of Alzheimer’s disease.
Understanding Context Dependent Relevance of Potential Cancer Drug Targ
Dr. Giulio Draetta, M.D., Ph.D., is a professor of Molecular and Cellular Oncology at The University of Texas MD Anderson Cancer Center. In this talk, given at the 2013 Signaling Pathways in Cancer Symposium sponsored by Cell Signaling Technology, Dr. Draetta discusses Understanding Context-Dependent Relevance of Potential Drug Targets. In this video, Dr. Draetta shares his thoughts on how our approach in thinking about and treating cancer needs to evolve in order to help patients with aggressive cancers like pancreatic cancer where the disease often relapses over time. Using an inducible KRAS model, Dr. Draetta describes how surviving cells treated with kinase inhibitors exhibit features of cancer stem cells and makes the link between the oncogenic KRAS driver mutation and the metabolic reprogramming of cancer cells.
Advances and Challenges for Understanding Macrophages in the Tumor Microenvironment
Exploring the plasticity of tumor-associated macrophages (TAMs) and challenges in distinguishing M1- versus M2-polarized macrophages. 👉Subscribe: http://youtube.com/user/cellsignaldotcom?sub_confirmation=1 Transcript: An important part of our immune system is its ability to differentiate between normal, healthy cells in the body, and those that are cancerous. The major problem with cancer cells is that they often find ways to avoid being attacked by the immune system. Often, this occurs when they increase the expression of immunosuppressive proteins in the tumor microenvironment, such as PD-1 and IDO. Over the past 15 years, therapeutics specifically blocking immunosuppressive proteins to activate T cells and the adaptive immune response have demonstrated therapeutic efficacy in treating cancer. Building on that success, therapeutics targeting the innate immune system, such macrophages and dendritic cells, are under pre-clinical and clinical investigation. Despite the unprecedented success of immunotherapy, scientists are still striving to do better. Not all patients respond to treatment, so scientists are trying to identify biomarkers that can predict whether a patient will respond to immunotherapy in advance. That way each person receives a personalized treatment strategy that is more likely to succeed. These so called predictive biomarkers could be the immunosuppressive proteins themselves, such as PD-L1, or it could be the accumulation of certain immunosuppressive cell types, such as tumor-associated macrophages also known as TAMs. Macrophages are critical to the development, maintenance, and also the eradication of cancer cells. In general, macrophages in the tumor microenvironment and at sites of wound healing can be polarized into two functional states: M1 or M2 macrophages. M1-polarized macrophages, referred to as Classically activated macrophages, are activated by cytokines such as interferon-γ, and they produce pro-inflammatory and immunostimulatory cytokines such as interleukin-12 and -23. Scientists have demonstrated M1 macrophages can have anti-tumoral properties, by scavenging and destroying phagocytosed tumor cells and stimulating helper T cell Type 1 responses. M2-polarized macrophages, also known as alternatively activated macrophages, are activated by cytokines such as interleukin-4, - 10, and -13. Most tumor-associated macrophages or TAMs are thought to resemble M2 macrophages. These cells play an important role in connecting inflammation with cancer. They express higher levels of anti-inflammatory cytokines, scavenging receptors, angiogenic factors, and proteases, than their M1-type polarized counterparts. TAMs can reprogram the immunosuppressive microenvironment and promote proliferation, invasion, and metastasis of tumor cells. They can stimulate tumor angiogenesis, and inhibit antitumor immune responses mediated by T cells. As the relationship between TAMs and malignant tumors becomes clearer, studies show they could be potential biomarkers for diagnosis, prognosis as well as therapeutic targets for cancer. For this reason, clear differentiation between M1 and M2 polarized macrophages is extremely important. In reality, M1 and M2 functional states exist as two extremes upon a continuum, with the balance being tipped one way or the other by higher or lower levels of cytokines in the tumor microenvironment. In addition, macrophages are plastic, therefore M1 macrophages, given the right stimuli in the right environment can become more M2-like, and vice versa. This makes differentiating these cell types very difficult. More than one marker must be used to identify M1 or M2 functional states, so multiplex labeling by flow cytometry, immunofluorescence, or immunohistochemistry is likely required. While a few proteins unique to each functional state have been identified, scientists continue to struggle to search for better, more specific markers delineating the two. Many of the proteins controlling the functional state of macrophages are intracellular making extracellular labeling insufficient, so complex protocols combining intracellular and extracellular detection simultaneously are necessary. If it wasn't complicated enough already, several of the key proteins involved in M1 versus M2 functional states differ between mouse models and humans, making the translation of biomarkers from pre-clinical research to clinical application sometimes impossible. It is an exciting and challenging time for immunologists, and there is so much more to be done in order to understand the role of innate immune cells, in particularly macrophages, in cancer as well as various other fields of disease research. Thanks for watching. Please subscribe to our channel for more videos and visit our website for more information. We'll see you next time, good luck with your experiments!
Oncoprotein activated feedback  biologic and therapeutic implications
Neal Rosen, M.D., Ph.D., Oncoprotein-activated feedback--biologic and therapeutic implications
Jeffrey Settleman, Ph.D., The Many Flavors of Resistance To Anti-Cancer Drugs
Jeffrey Settleman, Ph.D., the Senior Director of Discovery Oncology at Genentech, discussed his work investigating non-genetic/nonmutational drug-resistance to cancer therapeutics. "Understanding Signaling Pathways in Cancer", a symposium presented by the Koch Institute in partnership with Cell Signaling Technology, brought together speakers from leading cancer research organizations to present their latest findings on the signaling pathways leading to cancer.
PTMScan® Proteomics of Post-translational Modifications from Cell Signaling Technology
Mass spectrometry can be used to profile post-translationally modified (PTM) peptides but often requires samples enrichment in order to collect enough material for analysis. In this video, we discuss your different options using Cell Signaling Technology’s PTMScan technology, a proprietary proteomic method that combines antibody enrichment of PTM-containing peptides with LC-MS/MS. We’ll also take you through a real-life analysis example so you’ll see the type of information you’ll get. Cell Signaling Technology (CST) offers PTMScan services for that gives you information to help you identify novel post-translationally modified sites, identify and validate drug targets, understand drug mechanisms of action, and discover biomarkers from cells or tissue. PTMScan first starts with KinomeView analysis to help narrow down which motif antibodies to use. If you already have your motif antibody of interest picked out, you can skip directly to the second step – performing the PTMScan analysis. Currently, PTM service offering currently include phosphorylation, acetylation, and ubiquitination. Protease digested peptides are immunopurified using the motif antibody before samples are analyzed using LC tandem mass spectrometry to identify and quantify the PTMs in your sample. PTMScan service is a complete service that combines expertise in antibody technology, tandem mass spectrometry, and bioinformatics analysis to give you comprehensive and immediately relevant results.
Harnessing Cross Talk Between Signaling Pathways to Improve Cancer Tr
Michael B. Yaffe, Ph.D., M.D., Professor of Biology and Biological Engineering at MIT, discussed his work examining the use of multiple anti-cancer therapeutics to dynamically re-wire signaling networks.
Cellular Signal Transduction Animation: Protein Secretion and Vesicle Trafficking
Brief introduction to signal transduction featuring protein secretion and vesicle trafficking from mRNA translation to exocytosis. NOTE: There is no audio track with this video. Visit http://www.cellsignal.com for more information about Protein Secretion and Vesicle Trafficking.
The Study of Stem Cells - Part 3 of 3
Section Three - This learning video from Cell Signaling Technology, Inc. (CST) covers epigenetic regulators and marks commonly found during embryonic development. Visit http://www.cellsignal.com to see antibodies directed against stem cell and lineage markers.
Are There Differences in Validating Monoclonal vs. Polyclonal Antibodies?
A brief introduction to the advantages of monoclonal antibodies in terms of validation and reproducibility. Transcript The initial validation of...a new antibody, whether it's monoclonal or polyclonal, is the same. You are really looking at these for the same types of specificity [and] sensitivity you would hope for in an antibody. The real difference is more of a technical one. The biggest burden is on the vendor and that's the reproducibility. Monoclonals have significant advantages and that the reproducibility is significantly easier to achieve because of the monoclonal nature of it [and] the ability to manufacture it with greater consistency.
Chromatin crosslinking: how much time? Chromatin Immunoprecipitation (ChIP)  | CST Tech Tips
Learn how the optimal crosslinking time in Chromatin Immunoprecipitation (ChIP) can be influenced by sample type, and by target protein (histone, transcription factor, or cofactor). 👉Subscribe: http://youtube.com/user/cellsignaldotcom?sub_confirmation=1 👉CST Protocols and Troubleshooting: http://cellsignal.com/protocols 👉Get in touch with a CST scientist: http://cellsignal.com/support Transcript: - - What is the appropriate crosslinking time when performing ChIP? I'm Fang Chen, Senior Scientist in the ChIP group at Cell Signaling Technology, and this is CST Tech Tips. Crosslinking - or fixation - is the first step in the ChIP protocol. The purpose of crosslinking is to prevent dissociation of DNA from the protein during the 3 day ChIP experiment. Otherwise, the antibody can only pull down its target protein but not the DNA. Crosslinking is especially important for transcription factors and cofactors, because of their lower abundance and the weaker binding with DNA compared to histone proteins. Also, tissue is a more difficult sample type than cultured cells as it's harder for crosslinker to get into each single cell in tissue sample. Therefore, a longer crosslinking time can be helpful for transcription factors, cofactor and tissue materials. However, over-crosslinking leads to difficulties in chromatin shearing especially when using sonication to fragment chromatin DNA. So we need to find a balance where proteins of interest are firmly fixed with DNA and the chromatin remains shearable. Based on our experience, 10 minutes of crosslinking is enough for cultured cells, no matter what type of target the protein is: histone, transcription factor, or cofactor. For tissue samples, 10 minutes of crosslinking is adequate for histone protein ChIP, but an increased crosslinking time - up to 30 minutes - might be necessary for transcription factor and cofactor ChIP. I hope you found this video helpful. You can find full protocols for all applications on the specific product page at cellsignal.com. And if you have any questions about an antibody or a protocol, you can get in touch with one of our scientists at cellsignal.com/support. For more tech tips videos, subscribe to our channel, and we will see you next time. Good luck with your experiments! 👉About CST: Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high-quality research tools to the biomedical research community. Our employees operate worldwide from our U.S. headquarters in Massachusetts, and our offices in the Netherlands, China, and Japan. http://cellsignal.com/about
Epigenetic Analysis Using Highly Validated Antibodies and Optimized ChIP Assays
In this webinar, Dr. Sayura Aoyagi will present on important factors to consider when performing a ChIP assay, including use of highly validated antibodies, optimized protocols and reagents, and the advantages of using enzyme-based chromatin digestion over sonication-based chromatin fragmentation.
Yossi Shiloh, Ph D , ATM  regulating the cellular response to genotoxic stress, and more
Yossi Shiloh, Ph.D., Tel Aviv University Sackler School of Medicine
Towards robust and systematic translational systems for cancer therapeutic development
William Sellers, M.D., from the Novartis Institutes for BioMedical Research discusses research Towards robust and systematic translational systems for cancer therapeutic development
The Story of an Antibody, Whiteboard Edition
CST has always believed that the only way to produce and validate our products is to do it in-house. That way, we can guarantee that they are rooted in sound science, supported by the most rigorous testing and customer service, and that our antibodies will be specific and reproducible tools available to research scientists. To learn more about the antibody validation process at Cell Signaling Technology, visit us online at: https://www.cellsignal.com/common/content/content.jsp?id=ourApproach-validation-principles
Learn about PTMScan®
Antibody enrichment of post-translationally modified peptides for mass spectrometry-based proteomics. PTMScan® Proteomics from Cell Signaling Technology (CST)
The Study of Akt
This video provides in-depth information on upstream and downstream signaling mechanisms stemming from Akt and includes clinical ramifications of aberrant Akt signaling in causing disease (includes 3 sections)
Matrix Dependent Rewiring of Signaling Pathways Following Targeted Therapies
Joan Brugge, Ph.D., Chair of the Department of Cell Biology at Harvard Medical School, discussed her work examining pathways that allow for drug resistance of extracellular matrix-attached tumors.