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Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy

Authors: Anna Fuller
Anna FullerAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4810
, Aaron Wall
Aaron WallAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4811
, Michael D Crowther
Michael D CrowtherAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4812
, Angharad Lloyd
Angharad LloydAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4813
, Alexei Zhurov
Alexei ZhurovAffiliation: Cardiff University School of Dentistry, Heath Park, Cardiff, UK
Bio-protocol author page: a4814
, Andrew K. Sewell
Andrew K. SewellAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
Bio-protocol author page: a4815
, David K. Cole
David K. ColeAffiliation: Division of Infection and Immunity and Systems Immunity Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, UK
For correspondence: ColeDK@cf.ac.uk
Bio-protocol author page: a4816
 and Konrad Beck
Konrad BeckAffiliation: Cardiff University School of Dentistry, Heath Park, Cardiff, UK
For correspondence: BeckK@cf.ac.uk
Bio-protocol author page: a4799
,  date: 7/5/2017, 136 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2366.

[Abstract] T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability ...

Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen

Authors: Brent Veerman
Brent VeermanAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a4752
 and Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: ritu.chakravarti@utoledo.edu
Bio-protocol author page: a3737
,  date: 7/5/2017, 134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2367.

[Abstract] Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases (Wang et al., 2015). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed ...

Active Cdk5 Immunoprecipitation and Kinase Assay

Authors: Andrew N. Bankston
Andrew N. BankstonAffiliation 1: Department of Pharmacology, Emory University, Atlanta, GA
Affiliation 2: Department of Neurological Surgery, University of Louisville, Louisville, KY
Bio-protocol author page: a4746
, Li Ku
Li KuAffiliation: Department of Pharmacology, Emory University, Atlanta, GA
Bio-protocol author page: a4747
 and Yue Feng
Yue FengAffiliation: Department of Pharmacology, Emory University, Atlanta, GA
For correspondence: yfeng@emory.edu
Bio-protocol author page: a4748
,  date: 7/5/2017, 113 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2363.

[Abstract] Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et ...

Microvesicle Isolation from Rat Brain Extract Treated Human Mesenchymal Stem Cells

Authors: Ji Yong Lee
Ji Yong LeeAffiliation: Institute for BioMedical Convergence, Catholic Kwandong University-International St. Mary’s Hospital, Incheon-si, Republic of Korea
Bio-protocol author page: a4768
, Seong-Mi Choi
Seong-Mi ChoiAffiliation: Institute for BioMedical Convergence, Catholic Kwandong University-International St. Mary’s Hospital, Incheon-si, Republic of Korea
Bio-protocol author page: a4769
 and Han-Soo Kim
Han-Soo KimAffiliation: Department of Biomedical Sciences, College of Medical Convergence, Catholic Kwandong University, Gangneung-si, Gangwon-do, Republic of Korea
For correspondence: hankim63@gmail.com
Bio-protocol author page: a4770
,  date: 7/5/2017, 146 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2375.

[Abstract] Microvesicle (MVs) are submicron-sized membranous vesicles that are either actively released from cells via secretory compartments or shed from cell surface membranes. MVs are generated by many cell types and serve as vehicles that transfer biological information (e.g., protein, mRNA, and miRNA) to ...

Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth

Authors: Natalia V. Andreeva
Natalia V. AndreevaAffiliation: Laboratory of Stem and Progenitor Cell Biology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Bio-protocol author page: a4766
 and Alexander V. Belyavsky
Alexander V. BelyavskyAffiliation: Laboratory of Stem and Progenitor Cell Biology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
For correspondence: abelyavs@yahoo.com
Bio-protocol author page: a4767
,  date: 7/5/2017, 139 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2374.

[Abstract] Coating tissue culture vessels with the components of the extracellular matrix such as fibronectin and collagens provides a more natural environment for primary cells in vitro and stimulates their proliferation. However, the effects of such protein layers are usually rather modest, which might be explained ...

In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair

Authors: Melike Çağlayan
Melike ÇağlayanAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
Bio-protocol author page: a1849
 and Samuel H. Wilson
Samuel H. WilsonAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
For correspondence: wilson5@niehs.nih.gov
Bio-protocol author page: a1850
,  date: 6/20/2017, 238 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2341.

[Abstract] We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan et al., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture ...

Soft Agar Colony Formation Assay as a Hallmark of Carcinogenesis

Authors: Feng Du
Feng DuAffiliation: State key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, China
Bio-protocol author page: a4714
, Xiaodi Zhao
Xiaodi ZhaoAffiliation: State key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, China
For correspondence: leedyzhao@fmmu.edu.cn
Bio-protocol author page: a4715
 and Daiming Fan
Daiming FanAffiliation: State key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, China
Bio-protocol author page: a4716
,  date: 6/20/2017, 284 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2351.

[Abstract] Soft agar colony formation assay is established to estimate the anchorage-independent growth ability of cells. In this assay, a bottom layer of agar with complete media is poured and solidified first, followed by an upper layer containing a specified number of cells suspended in medium-agar mixture. ...

Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity

Authors: Trung M. Hoang
Trung M. HoangAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
Bio-protocol author page: a4648
, Tatiana Kent
Tatiana KentAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
Bio-protocol author page: a4649
 and Richard T. Pomerantz
Richard T. PomerantzAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
For correspondence: richard.pomerantz@temple.edu
Bio-protocol author page: a4650
,  date: 6/20/2017, 180 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2330.

[Abstract] DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and ...

Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System

Authors: Reka A. Haraszti
Reka A. HarasztiAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4671
, Andrew Coles
Andrew ColesAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4672
, Neil Aronin
Neil AroninAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4673
, Anastasia Khvorova
Anastasia KhvorovaAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4674
 and Marie-Cécile Didiot
Marie-Cécile DidiotAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
For correspondence: marie.didiot@umassmed.edu
Bio-protocol author page: a4675
,  date: 6/20/2017, 221 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2338.

[Abstract] Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and ...

DNA Fiber Assay upon Treatment with Ultraviolet Radiations

Authors: Alfano Luigi
Alfano LuigiAffiliation: Oncology Research Center of Mercogliano (CROM), Istituto Nazionale Per Lo Studio E La Cura Dei Tumori “Fondazione Giovanni Pascale”, IRCCS, Naples, Italy
Bio-protocol author page: a4568
, Antonio Giordano
Antonio GiordanoAffiliation: Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Department of Biology, Temple University, Philadelphia PA, USA
For correspondence: giordano@temple.edu
Bio-protocol author page: a4569
 and Francesca Pentimalli
Francesca PentimalliAffiliation: Oncology Research Center of Mercogliano (CROM), Istituto Nazionale Per Lo Studio E La Cura Dei Tumori “Fondazione Giovanni Pascale”, IRCCS, Naples, Italy
Bio-protocol author page: a4570
,  date: 6/5/2017, 317 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2301.

[Abstract] Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA damage Response (DDR). Ultra Violet (UV) rays are among the main environmental ...
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Scratch Wound Healing Assay

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
,  date: 3/5/2012, 57536 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.100.

[Abstract] The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. In a typical scratch wound healing assay, a “wound gap” in a cell monolayer ...

In vitro Culture of Human PBMCs

Authors: Santosh K Panda
Santosh K PandaAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
Bio-protocol author page: a221
 and Balachandran Ravindran
Balachandran RavindranAffiliation: Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India
For correspondence: ravindran8@gmail.com
Bio-protocol author page: a222
,  date: 2/5/2013, 45539 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.322.

[Abstract] Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by ...

Transwell Cell Migration Assay Using Human Breast Epithelial Cancer Cell

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
,  date: 2/20/2012, 44434 views, 7 Q&A
DOI: https://doi.org/10.21769/BioProtoc.99.

[Abstract] Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay is also useful in screens for compounds that act as chemoattractants or inhibitors of chemotaxis for cells. The assay employs a permeable layer of support, ...

Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)

Authors: Rabii Ameziane-El-Hassani
Rabii Ameziane-El-HassaniAffiliation 1: UBRM, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco
Affiliation 2: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Bio-protocol author page: a200
 and Corinne Dupuy
Corinne DupuyAffiliation 1: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Affiliation 2: University Paris, Sud 11, Orsay, France
For correspondence: dupuy@igr.fr
Bio-protocol author page: a201
,  date: 1/5/2013, 43878 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.313.

[Abstract] Reactive oxygen species (ROS) play a critical role in cellular physiopathology. ROS are implicated in cell proliferation, signaling pathways, oxidative defense mechanisms responsible for killing of bacteria, thyroid hormonosynthesis, etc. The cellular Redox homeostasis is balanced by oxidants and antioxidants ...

Clonogenic Assay

Author: Xiaodong Yang
Xiaodong YangAffiliation: Department of Neurology, University of California, San Francisco, USA
For correspondence: yangxiaodong1@yahoo.com
Bio-protocol author page: a43
,  date: 5/20/2012, 40592 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.187.

[Abstract] Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive ...

In vitro Human Umbilical Vein Endothelial Cells (HUVEC) Tube-formation Assay

Authors: Josephine MY Ko
Josephine MY KoAffiliation: Clinical Oncology Department, The University of Hong Kong, Hong Kong , Hong Kong SAR
Bio-protocol author page: a100
 and Maria Li Lung
Maria Li LungAffiliation: Clinical Oncology Department, The Univerisity of Hong Kong, Hong Kong, Hong Kong SAR
For correspondence: mlilung@hku.hk
Bio-protocol author page: a101
,  date: 9/20/2012, 33978 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.260.

[Abstract] Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. During angiogenesis, endothelial cells (ECs) undergo activation after binding of angiogenic factors to ...

[Bio101] Cell Adhesion Assay

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
,  date: 3/5/2012, 30222 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.98.

[Abstract] Cell adhesion, the binding of a cell to the extracellular matrix (ECM), other cells, or a specific surface, is essential for the growth and survival of the cell and also its communication with other cells. The process of cell adhesion involves a range of biological events such as three-dimensional re-organization ...

[Bio101] Calcium Phosphate Transfection of Eukaryotic Cells

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
,  date: 2/5/2012, 28873 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.86.

[Abstract] Transfection of DNA into cells is an indispensible protocol in molecular biology. While plenty of lipid-based transfection reagents are commercially available nowadays, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA ...

In vitro Tumorsphere Formation Assays

Authors: Sara Johnson
Sara JohnsonAffiliation: Biological Sciences Department, University of South Carolina, Columbia, USA
Bio-protocol author page: a224
, Hexin Chen
Hexin ChenAffiliation: Biological Sciences Department, University of South Carolina, Columbia, USA
For correspondence: hchen@biol.sc.edu
Bio-protocol author page: a225
 and Pang-Kuo Lo
Pang-Kuo LoAffiliation: Biological Sciences Department, University of South Carolina, Columbia, USA
Bio-protocol author page: a226
,  date: 2/5/2013, 25894 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.325.

[Abstract] A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. ...

Analysis of Protein Stability by the Cycloheximide Chase Assay

Authors: Shih-Han Kao
Shih-Han KaoAffiliation: Research Center for Tumor Medical Science, China Medical University, Taichung, Taiwan
Bio-protocol author page: a1920
, Wen-Lung Wang
Wen-Lung WangAffiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Bio-protocol author page: a1921
, Chi-Yuan Chen
Chi-Yuan ChenAffiliation: Department of Nutrition and Health Sciences, Chang Gung University of Science and Technology, Taoyuan, Taiwan
Bio-protocol author page: a1922
, Yih-Leong Chang
Yih-Leong ChangAffiliation: Department of Pathology and Graduate Institute of Pathology, National Taiwan University, Taipei, Taiwan
Bio-protocol author page: a1923
, Yi-Ying Wu
Yi-Ying WuAffiliation: Graduate Institute of Clinical Medicine, National Cheng Kung University, Taipei, Taiwan
Bio-protocol author page: a1924
, Yi-Ting Wang
Yi-Ting WangAffiliation 1: Chemical Biology and Molecular Biophysics Program, Academia Sinica, Taipei, Taiwan
Affiliation 2: Institute of Chemistry, Academia Sinica, Taipei, Taiwan
Affiliation 3: Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Bio-protocol author page: a1925
, Shu-Ping Wang
Shu-Ping WangAffiliation: Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, USA
Bio-protocol author page: a1926
, Alexey I Nesvizhskii
Alexey I NesvizhskiiAffiliation 1: Department of Pathology, University of Michigan, Ann Arbor, USA
Affiliation 2: Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, USA
Bio-protocol author page: a1927
, Yu-Ju Chen
Yu-Ju ChenAffiliation 1: Chemical Biology and Molecular Biophysics Program, Academia Sinica, Taipei, Taiwan
Affiliation 2: Institute of Chemistry, Academia Sinica, Taipei, Taiwan
Bio-protocol author page: a1928
, Tse-Ming Hong
Tse-Ming HongAffiliation: Graduate Institute of Clinical Medicine, National Cheng Kung University, Taipei, Taiwan
Bio-protocol author page: a1929
 and Pan-Chyr Yang
Pan-Chyr YangAffiliation 1: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Affiliation 2: Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
Affiliation 3: NTU Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan
Present address: Department of Internal Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
For correspondence: pcyang@ntu.edu.tw
Bio-protocol author page: a574
,  date: 1/5/2015, 24053 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1374.

[Abstract] Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity ...
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Mammalia
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