Biochemistry

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    Protocols in Current Issue
    Preparation of Yeast tRNA Sample for NMR Spectroscopy
    Authors:  Marjorie Catala, Alexandre Gato, Carine Tisné and Pierre Barraud, date: 06/20/2020, view: 448, Q&A: 0
    Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In ...
    Assessments of HLA-I Specificities of Anti-HLA-I Monoclonal Antibodies Using Solid Phase Bead Arrays
    Authors:  Anita J. Zaitouna, Daniel S. Ramon and Malini Raghavan, date: 06/20/2020, view: 436, Q&A: 0
    Human leukocyte antigen class I (HLA-I) molecules are a group of structurally-related cell surface proteins with a high degree of variability within the population. While only up to six variants are expressed in an individual person, the whole ...
    Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates
    Authors:  Joshua E. Mayfield, Seema Irani and Yan Zhang, date: 06/20/2020, view: 641, Q&A: 0
    Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive ...
    Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
    Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 313, Q&A: 0
    Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an ...
    Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme
    Authors:  Cintia Stival, Carolina Baro Graf, Pablo E. Visconti and Dario Krapf, date: 06/20/2020, view: 559, Q&A: 0
    In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell ...
    In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates
    Authors:  William L. Willis, Abigail Foster, Caitlin Henry, Lai Chu Wu and Wael Jarjour, date: 06/20/2020, view: 453, Q&A: 0
    Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic ...
    Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
    Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased ...
    Preparation of Yeast tRNA Sample for NMR Spectroscopy
    Authors:  Marjorie Catala, Alexandre Gato, Carine Tisné and Pierre Barraud, date: 06/20/2020, view: 448, Q&A: 0
    [Abstract] Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological ...
    Assessments of HLA-I Specificities of Anti-HLA-I Monoclonal Antibodies Using Solid Phase Bead Arrays
    Authors:  Anita J. Zaitouna, Daniel S. Ramon and Malini Raghavan, date: 06/20/2020, view: 436, Q&A: 0
    [Abstract] Human leukocyte antigen class I (HLA-I) molecules are a group of structurally-related cell surface proteins with a high degree of variability within the population. While only up to six variants are expressed in an individual person, the whole population contains thousands of different variants. The ability to distinguish specific variants is ...
    Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates
    Authors:  Joshua E. Mayfield, Seema Irani and Yan Zhang, date: 06/20/2020, view: 641, Q&A: 0
    [Abstract] Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques ...
    Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
    Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 313, Q&A: 0
    [Abstract] Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and ...
    Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme
    Authors:  Cintia Stival, Carolina Baro Graf, Pablo E. Visconti and Dario Krapf, date: 06/20/2020, view: 559, Q&A: 0
    [Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique ...
    In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates
    Authors:  William L. Willis, Abigail Foster, Caitlin Henry, Lai Chu Wu and Wael Jarjour, date: 06/20/2020, view: 453, Q&A: 0
    [Abstract] Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient ...
    Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
    [Abstract] Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane ...
    Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners
    Authors:  Anca F. Savulescu, Stoyan Stoychev, Sipho Mamputha and Musa M. Mhlanga, date: 06/05/2020, view: 799, Q&A: 0
    [Abstract] RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), ...
    Mouse Adipose Tissue Protein Extraction
    Authors:  Yu A. An and Philipp E. Scherer, date: 06/05/2020, view: 658, Q&A: 0
    [Abstract] As obesity becomes a global epidemic, the metabolism research field is increasingly focusing on studying the physiological and pathological roles of adipose tissues (AT). However, extracting proteins from AT is challenging due to abundant fat content of intracellular lipid droplets. Several commercial kits for extraction of AT proteins are ...
    Differential Fractionation of Erythrocytes Infected by Plasmodium berghei
    Authors:  Bénédicte Gnangnon, Véronique Peucelle and Christine Pierrot, date: 06/05/2020, view: 370, Q&A: 0
    [Abstract] The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected ...



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