scientificprotocols

Authors: Manish Kumar, Ruchi Verma & Gajendra Raghava 

Introduction

Prediction of mitochondrial proteins is one of the major challenge in the filed bioinformatics due to their importance living organism. Mitochondrial proteins are associated with diseases like Alzheimer, Perkinson and Type II diabetes. Thus it is important to develop method for predicting mitochondrial proteins. The existing subcellular localization methods predict most of the location with high accuracy except mitochondrial protein. In order to improve accuracy of prediction of mitochndrial protein we developed a novel method Mitpred, based on presence of exclusive mitochondrial domains.

Important points:

1. SVM models using split amino acid composition (25 N-terminal, 25 C-terminal, and remaining residues)

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Authors: Henrik H. Jensen , Holger B. Kramer & Benjamin G. Davis 

Introduction

Here we describe the efficient and facile synthesis of N-acetyl glucosamine propargyl O-glycoside, a compound which has been prepared previously by conventional methods. (1) In previous syntheses the glycoside was obtained as a mixture of anomers1a) and in moderate to acceptable yield1b). The described improved method exploits a high yielding and strongly β-selective lanthanide triflate catalysed (2) glycosylation reaction. In addition to reaction efficiency the method is characterised by its simplicity and facile compound isolation and purification.

The propargyl O-glycoside was prepared for its subsequent use in Cu(I) catalysed protein modification reactions. (3)

scientificprotocols

Authors: Pushpa Pandiyan 

Introduction

In vitro Treg suppression assays are performed to determine the functional effect of Treg cells on CD4 T cells. They are performed by co-culturing the responding population (Tresp) with the Treg cells or control CD4 cells (Tcon cells).

Reagents

1. Balb/C mice from which the needed cell populations are isolated.

• PBS/BSA (0.5% BSA)

scientificprotocols

Authors: Pushpa Pandiyan 

Introduction

The mouse model of inflammatory bowel disease (IBD) is a well-established system for studying Treg cell-mediated suppression in vivo (Asseman et al., 1999).

Procedure

Cell transfers in vivo

scientificprotocols

Authors: Harpreet Singh & Gajendra Raghava 

Introduction

There is tremendous change in strategy used for developing vaccine, over the years from whole pathogen to antigens and antigens to antigenic regions (epitopes). In subunit vaccine epitope is used instead of a complete protein as vaccine candidate. Thus prediction of epitopes particularly T-help epitopes is one of the major challenges in subunit vaccine design. It is well established that binding of a peptide to an MHC Class II molecule is a prerequisite for activation of antigen specific T-helper cells. In past number of methods have been developed for predicting MHC class II binders but these methods allow to predict binder for one or two MHC alleles. Thus not suitable for wide population as individual have limited number of MHC alleles. In order to overcome this problem we developed a method for predicting promiscuous MHC binders, which binds two or more than two alleles. This server will be useful for designing subunit vaccine effective fo

scientificprotocols

Authors: Sneh Lata, BK Sharma & Gajendra PS Raghava 

Introduction

Increase in the number of bacterial pathogens resistant to conventional antibiotics, with time, has prompted renewed interest in the use of alternative natural microbial inhibitors, antibacterial peptides (a subset of antimicrobial peptides). Antibacterial peptides are an evolutionarily conserved component of the innate immune response and are found among all classes of life i.e. both in vertebrates and invertebrates. These exhibit potent, broad spectrum activity which demonstrates them to be used as potential novel therapeutic agents. The experimental identification and designing of antibacterial peptides is a costly and time consuming affair. Thus, there is a need to develop computational tools for predicting this important class of peptides. So, a novel method AntiBP was developed in order to serve the purpose, which is first of its kind to do this. This method can predict whether a peptide is antibacterial or not with high accura

scientificprotocols

Authors: Carol Heckman, Sucheta Kanagasundaram, Marilyn Cayer & Jong Paige 

Introduction

Researchers whose work focuses on self-assembled structures in the biological sciences or nanostructures in the materials sciences are interested in high-resolution imaging. Certain self-assembled structures occupy a size range where the theoretical limit of resolution by light microscopy (LM), which is on the order of 160 nm, is not adequate to image them. The cell creates several types of protrusions. The filopodium (plural, filopodia) is a slender, tapering extension of cytoplasm with a mean width of 50-100 nm. Filopodia are especially challenging to visualize, because they may broaden to >160 nm at the base where they integrate with the rest of the cytoplasm. Although the broader parts of the structures are often used to estimate the prevalence of filopodia on cells, the greatest number of the structures may be missing from the image viewed by LM. Thus, LM imaging gives inaccurate and misleading results. To

scientificprotocols

Authors: Sudipto Saha & Gajendra Raghava 

Introduction

In present era use of genetically modified proteins in foods, therapeutics and biopharmaceuticals is increasing with exponential rate. Thus it is important to predict whether a modified protein allergenic or not. In 2003, the Codex Alimentarius Commission (Codex) conveyed a panel of international food safety regulators to review the FAO/ WHO 2001 recommendations and recognized the uncertainties associated with the bioinformatics part of the guidelines. They recommended various tests for examining allergenic behavior of proteins that includes source of gene, sequence similarities with known allergens, stability of protein and IgE bindings. Considering these points in mind a method was developed for predicting allergenic proteins, which is based on various approaches.

Reagents

Both formatted and non-formated sequences are accepted as input. For formatted sequences the server uses ReadSeq. software which can read most commonly used standar

scientificprotocols

Authors: Levi Ledgerwood & Jonathan Bromberg 

Introduction

Trafficking of lymphocytes through lymphatics and secondary lymphoid organs is crucial for immunity. While the process of T lymphocyte migration across high endothelial venules (HEV) into lymph nodes (LN) is well characterized, relatively little is known about the mechanisms that regulate migration of lymphocytes from tissues into afferent lymphatics. Several groups have attempted to develop in vitro methods to investigate lymphatic migration. Culture of freshly isolated lymphatic endothelial cells has proven very difficult, and ultimately no cell line produced has been shown to retain a lymphatic lineage after multiple rounds of passage. The C3H/HeJ mouse endothelial line SVEC4-10 is an SV-40 transformant of endothelial cells isolated from the axillary LN, and has been variously reported as lymphatic, HEV, or blood microvascular endothelium. We have characterized this cell line via flow cytometry, RT-PCR, and gene array analysis, and have

scientificprotocols

Authors: Wenwei Hu , Zhaohui Feng , Angelika Teresky & Arnold Levine 

Introduction

This protocol describes a procedure to search for novel p53 target genes. First, screen for potential p53 target gene using p53MH algorithm. Then, test these candidate genes using a series of molecular biological assays, including Chromatin immunoprecipitation assay, luciferase reporter activity assay, and regulation of the expression levels of mRNA and protein of these genes by p53 protein. This protocol also includes some assays to measure the mouse implantation and reproduction used in our study.

Reagents

1. Chromatin Immunoprecipitation (ChIP) Assay Kit (Catalog# 17-295, Millipore)

• Anti-p53 antibodies (anti-p53 polyclonal antibody FL393, Catalog# sc-6243; anti-human p53 monoclonal antibody DO-1, Catalog# sc-126, Santa Cruz Biotechnology)