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Analysis of Proteins by Immunoprecipitation
Laboratory Procedures, PJ Hansen Laboratory - University of Florida Analysis of Proteins by Immunoprecipitation P.J. Hansen1 1Dept. of Animal Sciences, University of Florida Introduction Immunoprecipitation is a procedure by which peptides or proteins that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics (molecular weight, isoelectric point, etc.). As usually practiced, the name of the procedure is a misnomer since removal of the antigen from solution does not depend upon the formation of an insoluble antibody-antigen complex. Rather, antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody (Figure 1). Thus, unlike other techniques based on immunoprecipitation, it is not necessary to determine the optimal antibody dilution that favors spontaneously-occurring immunoprecipitates. Figure 1. Schematic representation of the principle of immunoprecipitation. An antibody added to a mixture of radiolabeled (*) and unlabeled proteins binds specifically to its antigen (A) (left tube). Antibody- antigen complex is absorbed from solution through the addition of an immobilized antibody binding protein such as Protein A-Sepharose beads (middle panel). Upon centrifugation, the antibody-antigen complex is brought down in the pellet (right panel). Subsequent liberation of the antigen can be achieved by boiling the sample in the presence of SDS. Typically, the antigen is made radioactive before the immunoprecipitation procedure, either by culturing cells with radioactive precursor or by labeling the molecule after synthesis has been completed (e.g., by radioiodination to iodinate tyrosine residues or by sodium [3H]borohydride reduction to label carbohydrate). -
A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation Across Biological Membranes
De Castro Pereira, G., Allen, W., Watkins, D., Buddrus, L., Noone, D., Liu, X., Richardson, A., Collinson, I., & Chacinska, A. (2019). A high- resolution luminescent assay for rapid and continuous monitoring of protein translocation across biological membranes. Journal of Molecular Biology, 431(8), 1689-1699. https://doi.org/10.1016/j.jmb.2019.03.007 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1016/j.jmb.2019.03.007 Link to publication record in Explore Bristol Research PDF-document This is the final published version of the article (version of record). It first appeared online via Elsevier at https://doi.org/10.1016/j.jmb.2019.03.007 . Please refer to any applicable terms of use of the publisher. University of Bristol - Explore Bristol Research General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/ebr-terms/ Methods Notes A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes Gonçalo C. Pereira 1, William J. Allen 1, Daniel W. Watkins 1, Lisa Buddrus 1,2, Dylan Noone 1, Xia Liu 1, Andrew P. Richardson 1, Agnieszka Chacinska 3 and Ian Collinson 1,2 1 - School of Biochemistry, University of Bristol, Bristol, UK 2 - BrisSynBio, University of Bristol, Bristol, UK 3 - Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097, Warsaw, Poland Correspondence to Ian Collinson: School of Biochemistry, University Walk, University of Bristol, Bristol BS8 1TD, UK. -
The Immunoassay Guide to Successful Mass Spectrometry
The Immunoassay Guide to Successful Mass Spectrometry Orr Sharpe Robinson Lab SUMS User Meeting October 29, 2013 What is it ? Hey! Look at that! Something is reacting in here! I just wish I knew what it is! anti-phospho-Tyrosine Maybe we should mass spec it! Coffey GP et.al. 2009 JCS 22(3137-44) True or false 1. A big western blot band means I have a LOT of protein 2. One band = 1 protein Big band on Western blot Bands are affected mainly by: Antibody affinity to the antigen Number of available epitopes Remember: After the Ag-Ab interaction, you are amplifying the signal by using an enzyme linked to a secondary antibody. How many proteins are in a band? Human genome: 20,000 genes=100,000 proteins There are about 5000 different proteins, not including PTMs, in a given cell at a single time point. Huge dynamic range 2D-PAGE: about 1000 spots are visible. 1D-PAGE: about 60 -100 bands are visible - So, how many proteins are in my band? Separation is the key! Can you IP your protein of interest? Can you find other way to help with the separation? -Organelle enrichment -PTMs enrichment -Size enrichment Have you optimized your running conditions? Choose the right gel and the right running conditions! Immunoprecipitation, in theory Step 1: Create a complex between a desired protein (Antigen) and an Antibody Step 2: Pull down the complex and remove the unbound proteins Step 3: Elute your antigen and analyze Immunoprecipitation, in real life Flow through Wash Elution M 170kDa 130kDa 100kDa 70kDa 55kDa 40kDa 35kDa 25kDa Lung tissue lysate, IP with patient sera , Coomassie stain Rabinovitch and Robinson labs, unpublished data Optimizing immunoprecipitation You need: A good antibody that can IP The right beads: i. -
Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage
Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage by Griffin James Clausen B.S. Biomedical Engineering University of Minnesota – Twin Cities, 2012 Submitted to the Department of Biological Engineering in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Engineering at the Massachusetts Institute of Technology February 2019 © 2019 Massachusetts Institute of Technology. All rights reserved. Signature of Author: ____________________________________________________________ Griffin Clausen Department of Biological Engineering January 14, 2019 Certified by: ___________________________________________________________________ Angela Belcher James Mason Crafts Professor of Biological Engineering and Materials Science Thesis Supervisor Accepted by: ___________________________________________________________________ Forest White Professor of Biological Engineering Graduate Program Committee Chair This doctoral thesis has been examined by the following committee: Amy Keating Thesis Committee Chair Professor of Biology and Biological Engineering Massachusetts Institute of Technology Angela Belcher Thesis Supervisor James Mason Crafts Professor of Biological Engineering and Materials Science Massachusetts Institute of Technology Paul Blainey Core Member, Broad Institute Associate Professor of Biological Engineering Massachusetts Institute of Technology 2 Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage by Griffin James Clausen Submitted -
High-Throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity † ⊗ † ⊗ † ‡ † † § ∥ Yao Lu, , Jonathan J
Article pubs.acs.org/ac High-Throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity † ⊗ † ⊗ † ‡ † † § ∥ Yao Lu, , Jonathan J. Chen, , Luye Mu, , Qiong Xue, Yu Wu, Pei-Hsun Wu, Jie Li, ∥ † § † ⊥ Alexander O. Vortmeyer, Kathryn Miller-Jensen, Denis Wirtz, and Rong Fan*, , † Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06520, United States ‡ Department of Electrical Engineering, Yale University, New Haven, Connecticut 06520, United States § Department of Chemical and Biomolecular Engineering and the Johns Hopkins Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, Maryland 21218, United States ∥ Department of Pathology, Yale School of Medicine, New Haven, Connecticut 06520, United States ⊥ Yale Comprehensive Cancer Center, New Haven, Connecticut 06520, United States *S Supporting Information ABSTRACT: Secreted proteins dictate a range of cellular functions in human health and disease. Because of the high degree of cellular heterogeneity and, more importantly, polyfunctionality of individual cells, there is an unmet need to simultaneously measure an array of proteins from single cells and to rapidly assay a large number of single cells (more than 1000) in parallel. We describe a simple bioanalytical assay platform consisting of a large array of subnanoliter micro- chambers integrated with high-density antibody barcode microarrays for highly multiplexed protein detection from over a thousand single cells in parallel. This platform has been tested for both cell lines and complex biological samples such as primary cells from patients. We observed distinct heterogeneity among the single cell secretomic signatures that, for the first time, can be directly correlated to the cells’ physical behavior such as migration. Compared to the state-of-the-art protein secretion assay such as ELISpot and emerging microtechnology-enabled assays, our approach offers both high throughput and high multiplicity. -
Anti-GFP (Green Fluorescent Protein) Mab-Agarose Code No
D153-8 For Research Use Only. Page 1 of 2 Not for use in diagnostic procedures. MONOCLONAL ANTIBODY Anti-GFP (Green Fluorescent Protein) mAb-Agarose Code No. Clone Subclass Quantity D153-8 RQ2 Rat IgG2a Gel: 200 L BACKGROUND: Since the detection of intracellular 5) Dragone, L. L., et al., PNAS. 103, 18202-18207 (2006) [IP] Aequorea Victria Green Fluorescent Protein (GFP) requires 6) Darzacq, X., et al., J. Cell Biol. 173, 207-218 (2006) [ChIP] only irradiation by UV or blue light, it provides an excellent 7) Hayakawa, T., et al., Plant Cell Physiol. 47, 891-904 (2006) [IP] means for monitoring gene expression and protein 8) Obuse, C., et al., Nat. Cell Biol. 6, 1135-1141 (2004) [IP] localization in living cells. Agarose conjugated anti-GFP monoclonal antibody can detect GFP fusion protein on As this antibody is really famous all over the world, a lot of Immunoprecipitation. researches have been reported. These references are a part of such reports. SOURCE: This antibody was purified from hybridoma (clone RQ2) supernatant using protein G agarose. This INTENDED USE: hybridoma was established by fusion of mouse myeloma For Research Use Only. Not for use in diagnostic procedures. cell PAI with Wister rat lymphnode immunized with GFP purified from GFP expressed 293T cells by affinity 1 2 chromatographic technique using mouse anti-GFP. kDa 66 FORMULATION: 100 g of anti-GFP monoclonal antibody covalently coupled to 200 L of agarose gel and 45 provided as a 50% gel slurry suspended in PBS containing GFP fusion protein preservative (0.1% ProClin 150) for a total volume of 400 30 L. -
Strategies and Challenges for the Next Generation of Therapeutic Antibodies
FOCUS ON THERAPEUTIC ANTIBODIES PERSPECTIVES ‘validated targets’, either because prior anti- TIMELINE bodies have clearly shown proof of activity in humans (first-generation approved anti- Strategies and challenges for the bodies on the market for clinically validated targets) or because a vast literature exists next generation of therapeutic on the importance of these targets for the disease mechanism in both in vitro and in vivo pharmacological models (experi- antibodies mental validation; although this does not necessarily equate to clinical validation). Alain Beck, Thierry Wurch, Christian Bailly and Nathalie Corvaia Basically, the strategy consists of develop- ing new generations of antibodies specific Abstract | Antibodies and related products are the fastest growing class of for the same antigens but targeting other therapeutic agents. By analysing the regulatory approvals of IgG-based epitopes and/or triggering different mecha- biotherapeutic agents in the past 10 years, we can gain insights into the successful nisms of action (second- or third-generation strategies used by pharmaceutical companies so far to bring innovative drugs to antibodies, as discussed below) or even the market. Many challenges will have to be faced in the next decade to bring specific for the same epitopes but with only one improved property (‘me better’ antibod- more efficient and affordable antibody-based drugs to the clinic. Here, we ies). This validated approach has a high discuss strategies to select the best therapeutic antigen targets, to optimize the probability of success, but there are many structure of IgG antibodies and to design related or new structures with groups working on this class of target pro- additional functions. -
M.Sc. [Botany] 346 13
cover page as mentioned below: below: mentioned Youas arepage instructedcover the to updateupdate to the coverinstructed pageare asYou mentioned below: Increase the font size of the Course Name. Name. 1. IncreaseCourse the theof fontsize sizefont ofthe the CourseIncrease 1. Name. use the following as a header in the Cover Page. Page. Cover 2. the usein the followingheader a as as a headerfollowing the inuse the 2. Cover Page. ALAGAPPAUNIVERSITY UNIVERSITYALAGAPPA [Accredited with ’A+’ Grade by NAAC (CGPA:3.64) in the Third Cycle Cycle Third the in (CGPA:3.64) [AccreditedNAAC by withGrade ’A+’’A+’ Gradewith by NAAC[Accredited (CGPA:3.64) in the Third Cycle and Graded as Category–I University by MHRD-UGC] MHRD-UGC] by University and Category–I Graded as as Graded Category–I and University by MHRD-UGC] M.Sc. [Botany] 003 630 – KARAIKUDIKARAIKUDI – 630 003 346 13 EDUCATION DIRECTORATEDISTANCE OF OF DISTANCEDIRECTORATE EDUCATION BIOLOGICAL TECHNIQUES IN BOTANY I - Semester BOTANY IN TECHNIQUES BIOLOGICAL M.Sc. [Botany] 346 13 cover page as mentioned below: below: mentioned Youas arepage instructedcover the to updateupdate to the coverinstructed pageare asYou mentioned below: Increase the font size of the Course Name. Name. 1. IncreaseCourse the theof fontsize sizefont ofthe the CourseIncrease 1. Name. use the following as a header in the Cover Page. Page. Cover 2. the usein the followingheader a as as a headerfollowing the inuse the 2. Cover Page. ALAGAPPAUNIVERSITY UNIVERSITYALAGAPPA [Accredited with ’A+’ Grade by NAAC (CGPA:3.64) in the Third Cycle Cycle Third the in (CGPA:3.64) [AccreditedNAAC by withGrade ’A+’’A+’ Gradewith by NAAC[Accredited (CGPA:3.64) in the Third Cycle and Graded as Category–I University by MHRD-UGC] MHRD-UGC] by University and Category–I Graded as as Graded Category–I and University by MHRD-UGC] M.Sc. -
A Protein Secreted by the Salmonella Type III Secretion System Controls
RESEARCH ARTICLE A protein secreted by the Salmonella type III secretion system controls needle filament assembly Junya Kato1†, Supratim Dey2†, Jose E Soto1†, Carmen Butan1, Mason C Wilkinson2, Roberto N De Guzman2*, Jorge E Galan1* 1Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, United States; 2Department of Molecular Biosciences, University of Kansas, Lawrence, United States Abstract Type III protein secretion systems (T3SS) are encoded by several pathogenic or symbiotic bacteria. The central component of this nanomachine is the needle complex. Here we show in a Salmonella Typhimurium T3SS that assembly of the needle filament of this structure requires OrgC, a protein encoded within the T3SS gene cluster. Absence of OrgC results in significantly reduced number of needle substructures but does not affect needle length. We show that OrgC is secreted by the T3SS and that exogenous addition of OrgC can complement a DorgC mutation. We also show that OrgC interacts with the needle filament subunit PrgI and accelerates its polymerization into filaments in vitro. The structure of OrgC shows a novel fold with a shared topology with a domain from flagellar capping proteins. These findings identify a novel component of T3SS and provide new insight into the assembly of the type III secretion machine. DOI: https://doi.org/10.7554/eLife.35886.001 *For correspondence: [email protected] (RNDG); [email protected] (JEG) Introduction †These authors contributed Type III protein secretion systems (T3SSs) are highly conserved molecular machines encoded by equally to this work many gram-negative bacteria pathogenic or symbiotic to animals, plants, or insects (Gala´n et al., Competing interests: The 2014; Deng et al., 2017; Notti and Stebbins, 2016). -
Hiper® Immunoprecipitation Teaching Kit Is Stable for 12 Months from the Date of Manufacture Without Showing Any Reduction in Performance
U n z i p p i n g G e n e s P r o d u c t I n f o r m a t i o n ® HiPer Immunoprecipitation Teaching Kit Product Code: HTI016 Number of experiments that can be performed: 5 Duration of Experiment Storage Instructions The kit is stable for 12 months from the date of manufacture Store 5X Sample Loading Buffer and Protein Marker at -20oC Store 30% Acrylamide-Bisacrylamide Solution, 2.5X Tris SDS Buffer (pH 8.8), 5X Tris SDS Buffer (pH 6.8), 5X Tris-Glycine-SDS Gel Running Buffer, TEMED, Antigen, Antibody and Protein A Sepharose Beads at 2-8oC Other kit contents can be stored at room temperature (15-25oC) 1 Registered Office : Commercial Office 23, Vadhani Industrial Estate,LBS Marg, A-516, Swastik Disha Business Park, Tel: 00-91-22-6147 1919 15 WHO Mumbai - 400 086, India. Via Vadhani Indl. Est., LBS Marg, Fax: 6147 1920, 2500 5764 GMP Tel. : (022) 4017 9797 / 2500 1607 Mumbai - 400 086, India Email : [email protected] CERTIFIED Fax : (022) 2500 2286 Web : www.himedialabs.com The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is made or is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein Index Sr. No. Contents Page No. 1 Aim 3 2 Introduction 3 3 Principle 3 4 Kit Contents 4 5 Materials Required But Not Provided 4 6 Storage 4 7 Important Instructions 5 8 Procedure 5 9 Flowchart 6 10 Observation and Result 7 11 Interpretation 7 12 Troubleshooting Guide 8 12 SDS-PAGE 8 2 Aim: To learn the technique of immunoprecipitation which involves the precipitation of the antigen-antibody complex by Protein A beads Introduction: Immunoprecipitation (IP) is a widely used procedure in immunology where a protein or antigen is precipitated out of a solution using an antibody that specifically binds to that antigen. -
Supporting Information
Supporting Information Zhang et al. 10.1073/pnas.0811715106 SI Materials and Methods In Vitro Insulin Secretion Assay. INS-1 832/13 cells were seeded in Physical Studies. All blood glucose measurements were deter- 12-well culture plates after 72 h of siRNA treatment. After mined on whole venous blood by using an automated glucose washing twice with PBS, cells were starved for2hinstandard monitor (One Touch Basic, Lifescan) (1). Glucose tolerance assay buffer (SAB) (4). Subsequently, the medium was replaced tests were performed on mice after 16 h of fasting. Mice were by SAB containing 3 mM glucose (for basal secretion) or high injected intraperitoneally (i.p.) with D-glucose (2 g/kg of body glucose (15 mM) to measure insulin secretion during a 2-h weight) and blood was obtained at indicated time points. For incubation. Supernatants were removed to measure secreted insulin release, glucose (3 g/kg of body weight) was injected i.p., insulin, and insulin secretion results were normalized to cell and blood was collected at indicated time points (2). Serum number. The amount of insulin secretion was assayed by using an insulin levels were measured by ELISA with a rat insulin RIA kit (Linco Research). standard (Crystal Chem) (3). Statistical analysis was performed using a 2-tailed unpaired t test. ATP Determination. After 72 h of siRNA treatment, cells were starved and treated with low or high glucose and then lysed in Islet Isolation and Insulin Content Measurement. Mice were killed, lysis buffer (10 mM Tris, pH 7.5, 0.1 M NaCl, 1 mM EDTA, and the common bile duct was cannulated with a 27-mm needle in the 0.01% Triton X-100). -
Highly Multiplexed Profiling of Single-Cell Effector Functions Reveals
Highly multiplexed profiling of single-cell effector PNAS PLUS functions reveals deep functional heterogeneity in response to pathogenic ligands Yao Lua,1, Qiong Xuea,1, Markus R. Eiselea,b, Endah S. Sulistijoa, Kara Browerc, Lin Hana, El-ad David Amird, Dana Pe’erd, Kathryn Miller-Jensena,e,f,2, and Rong Fana,f,g,2 aDepartment of Biomedical Engineering, Yale University, New Haven, CT 06520; bInstitute for System Dynamics, University of Stuttgart, D-70563 Stuttgart, Germany; cIsoPlexis, New Haven, CT 06511; dDepartment of Biological Sciences, Columbia University, New York, NY 10027; eDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520; fYale Comprehensive Cancer Center, New Haven, CT 06520; and gYale Stem Cell Center, Yale School of Medicine, New Haven, CT 06520 Edited by Garry P. Nolan, Stanford University, Stanford, CA, and accepted by the Editorial Board January 12, 2015 (received for review September 1, 2014) Despite recent advances in single-cell genomic, transcriptional, functional heterogeneity has not been fully delineated due in part and mass-cytometric profiling, it remains a challenge to collect to the lack of technologies for quantifying all immune effector highly multiplexed measurements of secreted proteins from single functions at the level of single cells. cells for comprehensive analysis of functional states. Herein, we Previously, multiplex profiling of effector proteins in single cells combine spatial and spectral encoding with polydimethylsiloxane was limited (less than or equal to four) because of spectral overlap, (PDMS) microchambers for codetection of 42 immune effector for example, in a FLUOROSpot assay (8) or a nanowell-based proteins secreted from single cells, representing the highest multi- microengraving assay (9).