Journal of Biological Methods (JBM)
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163 research outputs found
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In vitro and in vivo model systems used in prostate cancer research
No full textNew incidence of prostate cancer is a major public health issue in the Western world, and has been rising in other areas of the globe in recent years. In an effort to understanding the molecular pathogenesis of this disease, numerous cell models have been developed, arising mostly from patient biopsies. The introduction of the genetically engineered mouse in biomedical research has allowed the development of murine models that allow for the investigation of tumorigenic and metastatic processes. Current challenges to the field include lack of an animal model that faithfully recapitulates bone metastasis of prostate cancer
Comprehensive analysis of signal transduction in three-dimensional ECM-based tumor cell cultures
No full textAnalysis of signal transduction and protein phosphorylation is fundamental to understand physiological and pathological cell behavior as well as identification of novel therapeutic targets. Despite the fact that more physiological three-dimensional cell culture assays are increasingly used, particularly proteomics and phosphoproteomics remain challenging due to easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates without compromising cell adhesion before cell lysis. The samples can be used for Western blotting as well as phosphoproteome array technology. This technique would be of interest for researchers working in all fields of biology and drug development
Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation
No full textReverse-transcription quantitative PCR (RT-qPCR) is a powerful technique for quantification of gene expression. Extraction of RNA directly from human and large animal intervertebral disc (IVD) tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. IVD is consisted of nucleus pulposus (NP) and annulus fibrosis (AF). We compared the yield and quality of RNA extracted from cultured cells using TRIzol and combined TRIzol-column method (TRIspin) with and without bovine NP tissue added for high proteoglycan content simulation. Higher yields were obtained using the TRIzol method compared to the TRIspin method and the quality of RNA extracted using both methods was comparable. Cryosectioning was by far the most effective homogenization method for the tough bovine NP tissue. The typical modification to TRIzol extraction by the use of high salt solution in a previously reported study for tissue with high proteoglycan was insufficient for bovine NP tissue, where undesirable precipitates were formed during the RNA precipitation step. This necessitates other modifications to the protocol. In our study, the combination of additional phase separation and high salt precipitation was shown to be effective to avoid the formation of the undesirable precipitates. The modified TRIzol method was compared with the TRIspin method and shown to give higher RNA yield which resulted in smaller Ct values in RT-qPCR. In addition, we showed that the cryosectioning method was applicable to bovine muscle, mouse IVD and rat NP tissues. This method of RNA preparation also allows simultaneous preparation of tissue for histological study and quantitative gene expression analysis
Deciphering hepatocellular responses to metabolic and oncogenic stress
No full textEach cell type responds uniquely to stress and fractionally contributes to global and tissue-specific stress responses. Hepatocytes, liver macrophages (MΦ), and sinusoidal endothelial cells (SEC) play functionally important and interdependent roles in adaptive processes such as obesity and tumor growth. Although these cell types demonstrate significant phenotypic and functional heterogeneity, their distinctions enabling disease-specific responses remain understudied. We developed a strategy for the simultaneous isolation and quantification of these liver cell types based on antigenic cell surface marker expression. To demonstrate the utility and applicability of this technique, we quantified liver cell-specific responses to high-fat diet (HFD) or diethylnitrosamine (DEN), a liver-specific carcinogen, and found that while there was only a marginal increase in hepatocyte number, MΦ and SEC populations were quantitatively increased. Global gene expression profiling of hepatocytes, MΦ and SEC identified characteristic gene signatures that define each cell type in their distinct physiological or pathological states. Integration of hepatic gene signatures with available human obesity and liver cancer microarray data provides further insight into the cell-specific responses to metabolic or oncogenic stress. Our data reveal unique gene expression patterns that serve as molecular “fingerprints” for the cell-centric responses to pathologic stimuli in the distinct microenvironment of the liver. The technical advance highlighted in this study provides an essential resource for assessing hepatic cell-specific contributions to metabolic and oncogenic stress, information that could unveil previously unappreciated molecular mechanisms for the cellular crosstalk that underlies the continuum from metabolic disruption to obesity and ultimately hepatic cancer
Isolation and quantification of N-glycans from immunoglobulin G antibodies for quantitative glycosylation analysis
No full textN-glycosylation is one of the critical quality attributes for the therapeutic monoclonal antibodies. Characterization of N-glycans of monoclonal antibodies provides valuable information about its therapeutic efficacy. We present a non-invasive method of isolating N-glycans from Immunoglobulin G based antibodies for glycosylation analysis. The method consists of purification of antibodies from biological solution and release of N-glycans with peptide-N-glycosidase F in a single consolidated process using a mini affinity ligand column (e.g. protein-A column). The method is highly reproducible with average coefficient of variation of 0.012 in the glycoform percentage distributions between the replicates. The method provides quantification of the molar yield of glycans as a function of molar concentration of antibody in a single analysis. To our knowledge, this is the first time this approach was used to detect and quantify any macro-heterogeneity of N-glycosylation in monoclonal antibody samples. This fairly rapid and very cost-efficient method would be of great interest for academic labs and biopharmaceutical industries
A quantitative, surface plasmon resonance-based approach to evaluating DNA binding by the c-Myc oncoprotein and its disruption by small molecule inhibitors
No full textThe use of small molecules to interfere with protein-protein interactions has tremendous therapeutic appeal and is an area of intense interest. Numerous techniques exist to assess these interactions and their disruption. Many, however, require large amounts of protein, do not allow interactions to be followed in real time, are technically demanding or require large capital expenditures and high levels of expertise. Surface plasmon resonance (SPR) represents a convenient alternative to these techniques with virtually none of their disadvantages. We have devised an SPR-based method that allows the heterodimeric association between the c-Myc (Myc) oncoprotein and its obligate partner Max to be quantified in a manner that agrees well with values obtained by other methods. We also adapted it to examine the ability of previously validated small molecules to interfere with Myc-Max heterodimerization and DNA binding. These inhibitors comprised two distinct classes of molecules that inhibit DNA binding by preventing Myc-Max interaction or distorting pre-formed heterodimers and rendering them incapable of DNA binding. Our studies also point out several potential artifacts and pitfalls to be considered when attempting to employ similar SPR-based methods. This technique should be readily adaptable to the study of other protein-protein interactions and their disruption by small molecules
Reliable and inexpensive expression of large, tagged, exogenous proteins in murine bone marrow-derived macrophages using a second generation lentiviral system
No full textOver the past two decades, researchers have struggled to efficiently express foreign DNA in primary macrophages, impeding research progress. The applications of lipofection, electroporation, microinjection, and viral-mediated transfer typically result in disruptions in macrophage differentiation and function, low expression levels of exogenous proteins, limited efficiency and high cell mortality. In this report, after extensive optimization, we present a method of expressing large tagged proteins at high efficiency, consistency, and low cost using lentiviral infection. This method utilizes laboratory-propagated second generation plasmids to produce efficient virus that can be stored for later use. The expression of proteins up to 150 kDa in size is achieved in 30–70% of cells while maintaining normal macrophage differentiation and morphology as determined by fluorescence microscopy and Western blot analysis. This manuscript delineates the reagents and methods used to produce lentivirus to express exogenous DNA in murine bone marrow-derived macrophages sufficient for single cell microscopy as well as functional assays requiring large numbers of murine bone marrow-derived macrophages
Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality
No full textMicrospheres are often used as handles for protein purification or force spectroscopy. For example, optical tweezers apply forces on trapped particles to which motor proteins are attached. However, even though many attachment strategies exist, procedures are often limited to a particular biomolecule and prone to non-specific protein or surface attachment. Such interactions may lead to loss of protein functionality or microsphere clustering. Here, we describe a versatile coupling procedure for GFP-tagged proteins via a polyethylene glycol linker preserving the functionality of the coupled proteins. The procedure combines well-established protocols, is highly reproducible, reliable, and can be used for a large variety of proteins. The coupling is efficient and can be tuned to the desired microsphere-to-protein ratio. Moreover, microspheres hardly cluster or adhere to surfaces. Furthermore, the procedure can be adapted to different tags providing flexibility and a promising attachment strategy for any tagged protein
Enzymatic processes in alternative reaction media: a mini review
No full textBiocatalysis is a growing field in the production of fine chemicals and will most probably increase its share in the future. Enzymatic reactions are carried out under mild conditions, i.e., non-toxic solvents, low temperature and pressure, which eliminates most environmental drawbacks associated with conventional production methods. The superiority of chemo-, regio- and enantioselectivity of enzymes exhibit significant advantages over conventional catalysts for production of fine chemicals, flavors, fragrances, agrochemicals and pharmaceuticals. Enzymes can function both in aqueous and non-aqueous solvents. As a result of the growing scientific and industrial interest towards green chemistry, green solvent systems, which are mainly water, supercritical fluids, ionic liquids, fluorinated solvents, and solvent-free systems have become more popular in biocatalysis. However, the activity and selectivity of an enzyme is heavily dependent on solvent properties. In this review, various green solvents were classified and some of their influential features on enzyme activity were discussed
Open source and DIY hardware for DNA nanotechnology labs
No full textA set of instruments and specialized equipment is necessary to equip a laboratory to work with DNA. Reducing the barrier to entry for DNA manipulation should enable and encourage new labs to enter the field. We present three examples of open source/DIY technology with significantly reduced costs relative to commercial equipment. This includes a gel scanner, a horizontal PAGE gel mold, and a homogenizer for generating DNA-coated particles. The overall cost savings obtained by using open source/DIY equipment was between 50 and 90%