Current Protocols in Pharmacology最新文献

{"title":"Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3","authors":"Elizabeth A. Caine,&nbsp;Sarah D. Mahan,&nbsp;Rebecca L. Johnson,&nbsp;Amanda N. Nieman,&nbsp;Ngan Lam,&nbsp;Curtis R. Warren,&nbsp;Kristin M. Riching,&nbsp;Marjeta Urh,&nbsp;Danette L. Daniels","doi":"10.1002/cpph.81","DOIUrl":"10.1002/cpph.81","url":null,"abstract":"<p>To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live-cell knockout strategy that is applicable to numerous proteins. This modular protein-tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag-mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence-Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11-amino-acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG-13, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. © 2020 The Authors.</p><p><b>Basic Protocol 1</b>: CRISPR/Cas9 insertion of HaloTag or HiBiT-HaloTag</p><p><b>Basic Protocol 2</b>: HaloPROTAC3 degradation of endogenous HaloTag fusions</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.81","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38725727","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preclinical Models for Studying the Impact of Macrophages on Cancer Cachexia","authors":"Spas Dimitrov Markov,&nbsp;Daisy Gonzalez,&nbsp;Kamiya Mehla","doi":"10.1002/cpph.80","DOIUrl":"10.1002/cpph.80","url":null,"abstract":"<p>Cancer-associated cachexia is defined by loss of weight and muscle mass, and by the potential loss of adipose tissue accompanied by insulin resistance and increased resting energy expenditure. Cachexia is most prevalent in pancreatic cancer, the third leading cause of cancer-related deaths. While various factors interact to induce cachexia, the precise mechanisms underlying this clinical condition are not fully understood. Clinically relevant animal models of cachexia are needed given the lack of standard diagnostic methods or treatments for this condition. Described in this article are in vitro and in vivo models used to study the role of macrophages in the induction of cachexia in pancreatic cancer. Included are procedures for isolating and culturing bone marrow−derived macrophages, harvesting tumor- and macrophage-derived conditioned medium, and studying the effect of conditioned medium on C2C12 myotubes. Also described are procedures involving the use of an orthotopic model of pancreatic cancer, including a method for examining skeletal muscle atrophy in this model. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: In vitro model of pancreatic tumor-induced cachexia using C2C12 cell lines (myotube model)</p><p><b>Support Protocol 1</b>: Molecular evaluation of cachectic markers in C2C12 myotubes using real-time PCR and immunoblotting</p><p><b>Basic Protocol 2</b>: In vivo model to study cachectic phenotype in pancreatic tumor-bearing mice</p><p><b>Support Protocol 2</b>: Evaluation of cachectic markers in the skeletal muscle of tumor-bearing mice</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.80","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38662761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Vitro Permeation Test (IVPT) for Pharmacokinetic Assessment of Topical Dermatological Formulations","authors":"Leandro L. Santos,&nbsp;Nathaniel J. Swofford,&nbsp;Brandon G. Santiago","doi":"10.1002/cpph.79","DOIUrl":"10.1002/cpph.79","url":null,"abstract":"<p>In vitro assessment of topical (dermal) pharmacokinetics is a critical aspect of the drug development process for semi-solid products (e.g., solutions, foams, sprays, creams, gels, lotions, ointments), allowing for informed selection of new chemical entities, optimization of prototype formulations during the nonclinical stage, and determination of bioequivalence of generics. It can also serve as a tool to further understand the impact of different excipients on drug delivery, product quality, and formulation microstructure when used in parallel with other techniques, such as analyses of rheology, viscosity, microscopic characteristics, release rate, particle size, and oil droplet size distribution. The in vitro permeation test (IVPT), also known as in vitro skin penetration/permeation test, typically uses ex vivo human skin in conjunction with diffusion cells, such as Franz (or vertical) or Bronaugh (or flow-through) diffusion cells, and is the technique of choice for dermal pharmacokinetics assessment. Successful execution of the IVPT also involves the development and use of fit-for-purpose bioanalytical methods and procedures. The protocols described herein provide detailed steps for execution of the IVPT utilizing flow-through diffusion cells and for key aspects of the development of a liquid chromatography–tandem mass spectrometry method intended for analysis of the generated samples (epidermis, dermis, and receptor solution). © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: In vitro permeation test</p><p><b>Support Protocol</b>: Dermatoming of ex vivo human skin</p><p><b>Basic Protocol 2</b>: Bioanalytical methodology in the context of the in vitro permeation test</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"91 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.79","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38435774","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Localized Aldara (5% Imiquimod)–Induced Psoriasiform Dermatitis Model in Mice Using Finn Chambers","authors":"Szabina Horváth,&nbsp;Ágnes Kemény,&nbsp;Erika Pintér,&nbsp;Rolland Gyulai","doi":"10.1002/cpph.78","DOIUrl":"10.1002/cpph.78","url":null,"abstract":"<p>The expanding number of research studies utilizing the imiquimod-induced psoriasiform dermatitis model attests to the usefulness of this procedure. Advantages of this model include rapid development of the skin response and cost-effectiveness. A major limitation is that application of imiquimod cream over large areas of skin, as well as licking and ingestion of the cream, may lead to severe systemic inflammation, which can cause a general decline in health, splenomegaly, and death. In this protocol, Finn chambers are used to localize the imiquimod cream to a small area of the skin. This results in production of severe and reproducible psoriatic skin reactions with significantly less imiquimod, greatly reducing the possibility of untoward systemic effects. Moreover, having psoriasiform and control skin areas on the same mice decreases inter-animal differences. The protocol can be readily adapted for other skin disease models involving topical application of test agents. This article also details functional measurements performed during assays, including skin thickness, blood perfusion, semiquantitative histopathological evaluation, determination of scaling score to monitor psoriatic symptoms, and collection of spleen and body weight data to identify systemic effects. © 2020 The Authors.</p><p><b>Basic Protocol</b>: Use of Finn chambers to induce psoriasiform skin reactions with imiquimod</p><p><b>Support Protocol 1</b>: Measurement of double-fold dorsal skin thickness</p><p><b>Support Protocol 2</b>: Measurement of blood perfusion</p><p><b>Support Protocol 3</b>: Determination of scaling score</p><p><b>Support Protocol 4</b>: Semiquantitative histopathological scoring</p><p><b>Support Protocol 5</b>: Assessment of systemic side effects in response to imiquimod application</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"90 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.78","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38259413","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Autologously Humanized Mice for Immune-Oncologic Studies","authors":"Juan Fu,&nbsp;Young J. Kim","doi":"10.1002/cpph.76","DOIUrl":"10.1002/cpph.76","url":null,"abstract":"<p>With the rapid approval of immune checkpoint inhibitors for lung, melanoma, breast, genitourinary, and hematological malignancies, the hematopoietic cells in the tumor microenvironment (TME) are now considered an important, if not essential, consideration for cancer scientists. In many instances, syngeneic murine models have not been highly predictive for responsiveness in clinical trials. Our limited understanding of the human TME have, therefore, precluded a rational translation of immunotherapeutic combinations. This has led to the adoption of hematopoietic humanized murine models for the study of human tumor immunology in vivo. However, concerns about chimerism rates, HLA mismatching, and incomplete reconstitution of the innate immune system have driven a quest for improvements in these allogeneic humanized murine systems. Presented in this article is a completely autologous xenotransplantation method for reconstituting the human tumor immune microenvironment in vivo without the use of a patient's peripheral blood which is known to be associated with low engraftment rates. With this new approach, the current limitations of allogeneic humanized models are avoided by using matched bone marrow cells (BMCs) and derived tumor xenoplants (PDXs) from solid tumors in cancer patients. This autologous system provides a platform for studying endogenous lymphocytic and myeloid cell infiltration into the human tumor in vivo. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Autologous reconstitution of human tumors</p><p><b>Support Protocol 1</b>: Transduction of BMCs and/or tumor cells prior to autologous reconstitution</p><p><b>Support Protocol 2</b>: Modeling immunotherapeutic agents in an autologously humanized model</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.76","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37985881","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment of Humanized Mice from Peripheral Blood Mononuclear Cells or Cord Blood CD34+ Hematopoietic Stem Cells for Immune-Oncology Studies Evaluating New Therapeutic Agents","authors":"Bhavna Verma,&nbsp;Amy Wesa","doi":"10.1002/cpph.77","DOIUrl":"10.1002/cpph.77","url":null,"abstract":"The clinical success of immune checkpoint modulators and the development of next‐generation immune‐oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdcscid Il2rgtm1Sug) and triple‐transgenic NOG‐EXL [Prkdcscid Il2rgtm1Sug Tg (SV40/HTLV‐IL3, CSF2)], which expresses human IL‐3 and GM‐CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post‐transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T‐cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC‐HIS model can be limited due to onset of xenogeneic graft‐versus‐host disease (xGVHD) within 3 to 5 weeks post‐implantation. Host deficiency in MHC class I, as occurs in beta‐2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood–derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC‐HIS and PBMC‐HIS mouse models are provided. In addition, the co‐engraftment and growth kinetics of patient‐derived and cell line–derived xenograft tumors in humanized mice and recovery of tumor‐infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors.","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.77","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37973440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Screening Assay Protocols Targeting the Nav1.7 Channel Using Qube High-Throughput Automated Patch-Clamp System","authors":"Binbin Qian,&nbsp;Sung Hoon Park,&nbsp;Weifeng Yu","doi":"10.1002/cpph.74","DOIUrl":"10.1002/cpph.74","url":null,"abstract":"<p>This article describes the basic procedure for setting up the screening protocol and recording data for Nav1.7 on a Qube automated patch-clamp system. Three protocols along with step-by-step details are provided. First, we describe a protocol to estimate <i>V</i>_half, the voltage at which half of the channels are inactivated, using traditional steady-state inactivation measurement as well as a new adaptive online estimation. Second, we establish a state-dependent protocol using adaptive online <i>V</i>_half measurement to obtain a concentration response curve (CRC) on known reference blockers. Last, we introduce a use-dependent protocol. In our hands, the sample reference demonstrated good state- and use-dependent inhibition of Nav1.7. © 2020 by John Wiley &amp; Sons, Inc.</p><p><b>Basic Protocol 1</b>: Comparing <i>V</i>_half measurements using offline calculation and a new adaptive online estimation</p><p><b>Basic Protocol 2</b>: Concentration response experiments using a state-dependent protocol</p><p><b>Basic Protocol 3</b>: Concentration response experiments using a use-dependent protocol</p><p><b>Support Protocol</b>: Cell culture protocol</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.74","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37826255","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Electrophysiological Studies of GABAA Receptors Using QPatch II, the Next Generation of Automated Patch-Clamp Instruments","authors":"Melanie Schupp,&nbsp;Sung Hoon Park,&nbsp;Binbin Qian,&nbsp;Weifeng Yu","doi":"10.1002/cpph.75","DOIUrl":"10.1002/cpph.75","url":null,"abstract":"<p>Ligand-gated ion channels (LGICs) are a group of diverse ion channels that are gated by ligands and play important roles in normal physiological and pathological conditions. Many of them are drug targets that have been pursued, are being pursued, and will likely be pursued in the future by pharmaceutical companies and academic groups for a variety of diseases. One of those LGICs is the GABA_A receptor, a heterooligomeric chloride channel that can be blocked and modulated at various sites. In order to study the receptor's functional response to compounds, the manual patch-clamp method provides a detailed but low-throughput electrophysiological characterization. QPatch II, a next-generation automated patch clamp machine that was recently developed by Sophion Bioscience, provides an automated electrophysiological study of ion channels. In this article, we use the GABA_A receptor as an example for studying LGICs and describe two detailed protocols for using QPatch II to carry out pharmacological studies on the receptor. © 2020 by John Wiley &amp; Sons, Inc.</p><p><b>Basic Protocol 1</b>: Ligand concentration-response experiment (GABA_A receptor) on QPatch II</p><p><b>Alternate Protocol</b>: Non-cumulative ligand concentration-response experiment (GABA_A receptor) on QPatch II</p><p><b>Support Protocol 1</b>: Cell culture of HEK293-hGABA_A (α5β3γ2)</p><p><b>Support Protocol 2</b>: Data analysis for Basic Protocol 1</p><p><b>Support Protocol 3</b>: Data analysis for Alternate Protocol</p><p><b>Basic Protocol 2</b>: Antagonist dose-response experiment (GABA_A receptor) on QPatch II</p><p><b>Support Protocol 3</b>: Data analysis for Basic Protocol 2</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"89 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.75","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37817448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Automated Dynamic Clamp for Simulation of I_K1 in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Real Time Using Patchliner Dynamite⁸.","authors":"Nadine Becker,&nbsp;András Horváth,&nbsp;Teun De Boer,&nbsp;Alan Fabbri,&nbsp;Christian Grad,&nbsp;Niels Fertig,&nbsp;Michael George,&nbsp;Alison Obergrussberger","doi":"10.1002/cpph.70","DOIUrl":"https://doi.org/10.1002/cpph.70","url":null,"abstract":"<p><p>Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high-throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC-CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1-2.3), which conduct the cardiac inward rectifier potassium current (I_K1 ). I_K1 is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC-CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., I_K1 ) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC-CMs. In this article, we describe the method of using hiPSC-CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add-on (Dynamite⁸ ). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell-derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated I_K1 and compensate seal resistance Support Protocol 1: Cardiomyocyte plating and culture Support Protocol 2: Cell harvesting and dissociation Alternate Protocol: Recording action potential pharmacology at physiologic temperatures.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"88 1","pages":"e70"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.70","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37484130","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorescent Western Blotting: High Sensitivity Detection of Multiple Targets.","authors":"Tom Berkelman","doi":"10.1002/cpph.72","DOIUrl":"https://doi.org/10.1002/cpph.72","url":null,"abstract":"<p><p>Western blotting with fluorescence detection offers the possibility of detecting multiple targets simultaneously on a single blot. Primary antibodies are increasingly available from multiple hosts, and there are now a wide variety of dye labels to exploit multiple imaging channels. If primary and secondary antibodies are selected so that individual targets can be discriminated, multiple antigens can be detected and quantified in a single experiment. Current fluorescence imaging instrumentation offers multiple detection channels and gives sensitivity comparable to other methods. The method described in this article allows multiple targets to be quantified simultaneously and reduces the need for stripping and re-probing. It also allows loading controls to be detected alongside the targets of interest. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Five-plex western blot detection, including tubulin detection for loading control.</p>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"88 1","pages":"e72"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.72","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37555255","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}