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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Purification of a Recombinant Protein with Cellulose-Binding Module 3 as the Affinity Tag
Easy-to-perform and low-cost protein purification methods are in high demand for the mass production of commonly used enzymes that play an important role in bioeconomy. A low-cost and rapid recombinant protein purification system was developed using CBM3 (family 3 cellulose-binding module) as affinity tag. This protocol describes the purification of CBM3-fusion protein and tag-free protein expressed in Pichia pastoris using CBM3 as an affinity tag. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Affinity Purification of Heme-Tagged Proteins
Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an l-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedu...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
1D4: A Versatile Epitope Tag for the Purification and Characterization of Expressed Membrane and Soluble Proteins
Incorporation of short epitope tags into proteins for recognition by commercially available monoclonal or polyclonal antibodies has greatly facilitated the detection, characterization, localization, and purification of heterologously expressed proteins for structure–function studies. A number of tags have been developed, but many epitope–antibody combinations do not work effectively for all immunochemical techniques due to the nature of the tag and the specificity of the antibodies. A highly versatile, multipurpose epitope tag is the 9 amino acid C-terminal 1D4 peptide. This peptide tag together with the Rho1D4...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Bimolecular Affinity Purification: A Variation of TAP with Multiple Applications
The identification of true interacting partners of any given bait can be plagued by the nonspecific purification of irrelevant proteins. To avoid this problem, Tandem Affinity Purification (TAP) is a widely used procedure in molecular biology as this reduces the chance of nonspecific proteins being present in the final preparation. In this approach, two different affinity tags are fused to the protein bait. Herein, we review in detail a variation on the TAP procedure that we have previously developed, where the affinity moieties are placed on two different proteins that form a complex in vivo. This variation, which we refe...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
An Efficient Fluorescent Protein-Based Multifunctional Affinity Purification Approach in Mammalian Cells
Knowledge of an individual protein’s modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501–511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression leve...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Targeted Purification of SnAvi-Tagged Proteins
Tandem affinity purification (TAP) is a powerful technique to identify protein complex members. The modular composition of TAP-tags allows two sequential protein enrichment steps and thereby drastically reduces the amount of contaminants. Here, we describe the application of the SnAvi-tag—a TAP-tag useful in different expression systems. Due to its modular composition, this tag is multifunctional and facilitates among others the in vivo visualization of tagged proteins and their cell type specific activation. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Purification of Recombinant Proteins with a Multifunctional GFP Tag
Green fluorescent protein (GFP) is the most widespread fluorescent reporter for cellular localization and interaction of proteins. Because GFP itself is not the protein purification tag, protein purification is generally carried out with the aid of additional affinity tags. We have recently engineered a “multifunctional GFP” (mfGFP), a variant of enhanced GFP (EGFP), in which multiple affinity tags are inserted in tandem into an internal loop of EGFP. The mfGFP can be used as a fluorescent reporter and an affinity tag, and is compatible with various expression systems in prokaryotic and eukaryotic cells. Herein...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
An Improved In Vivo Biotinylation Strategy Combined with FLAG and Antibody Based Approaches for Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells
The proteome in mouse embryonic stem cells has not been extensively studied in comparison to other cellular systems, limiting our understanding of multi-protein complex functions in stem cell biology. Several affinity purification techniques followed by mass spectrometry analysis have been designed and validated to identify protein–protein interaction networks. One such approach relies on in vivo biotinylation of a protein of interest and subsequent pull-down of its interacting partners using streptavidin-conjugated agarose beads. This technique takes advantage of the high affinity between biotin and streptavidin, al...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Detection of Protein–Protein Interactions Using Tandem Affinity Purification
Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for s...
Source: Springer protocols feed by Protein Science - June 19, 2014 Category: Biochemistry Source Type: news
Preparation of Heteroelement-Incorporated and Stable Isotope-Labeled Protein Standards for Quantitative Proteomics
A major obstacle for further development of quantitative proteomics is the lack of accurately quantified protein standards. The following protocol describes innovative methods for the production of stable isotope-labeled protein standards. Their production is achieved by cell-free protein synthesis, which enables simultaneous incorporation of selenomethionine and stable isotope-labeled amino acids. The selenium tag allows sensitive and accurate quantification by inductively coupled plasma mass spectrometry (ICP-MS). The stable isotope label allows internal standardization in mass spectrometry-based proteomics by electrospr...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
The Secretome Analysis by High-Throughput Proteomics and Multiple Reaction Monitoring (MRM)
The secretome is a sub-proteome of great interest in several fields of biomedical sciences, especially as a source of diagnostics and therapeutic targets. Proteomics has been contributing significantly to elucidate the secretome of a great diversity of cells, tissues, and organisms, turning profiles of thousands of proteins a usual practice. After elucidation of long protein lists, targeted proteomics also plays important roles in accurate quantification and validation of such secreted proteins. Here we present detailed protocols to explore and quantify the secretome of cancer cells, even though this protocol can be employ...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Use of Universal Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-Based Selected Reaction Monitoring (SRM) Approach for Verification of Breast Cancer-Related Protein Markers
Mass spectrometry-based proteomics facilitates high-throughput discovery of protein markers for diagnosis and treatment of breast cancer patients. Hundreds of putative prognostic and predictive markers are being identified every year, but only a very small proportion of them can be validated as clinically relevant markers. A quantitative and cost-efficient verification method is highly desirable to pick up real “nuggets” from the “sand.” To fulfill these criteria, we previously introduced a stable isotope labeling by amino acids in cell culture (SILAC)-based selected reaction monitoring (SRM) approa...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Biomarker Verification Using Selected Reaction Monitoring and Shotgun Proteomics
Shotgun proteomics (liquid chromatography-electrospray ionization-mass spectrometry, LC-ESI-MS/MS) has dominated the strategies for global protein expression in subcells, cells, tissues, and whole organisms with several types of approaches, as isobaric tags for relative and absolute quantification (iTRAQ), isotope-coded affinity tags (ICAT), or stable isotope labeling using amino acids in cell culture (SILAC) and non-labeling (label free) methods. Shotgun proteomics practically replaced the classical 2D gel electrophoresis. Selected reaction monitoring (SRM), also denominated multiple reaction monitoring (MRM), is a target...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Mapping Protein Complexes Using Covalently Linked Antibodies and Isobaric Mass Tags
Affinity enrichment techniques in combination with quantitative proteomics enable the unbiased identification of protein–protein interaction, and thus the delineation of protein complexes and interaction networks. Here, we describe an immunoaffinity enrichment approach that employs covalently immobilized antibodies for the identification of protein–protein interactions of endogenously expressed proteins under near-to-physiological conditions. Specifically enriched proteins are identified using shotgun mass spectrometry and isobaric mass tag-based relative quantification. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
Application of Shotgun Proteomics for Discovery-Driven Protein–Protein Interaction
Affinity purification of protein complexes and identification of co-purified proteins by mass spectrometry is a powerful method to discover novel protein–protein interactions. Application of this method to the study of biological systems often requires the ability to process a large number of samples. Hence, there is great need to generate proteomic workflows compatible with large-scale studies. The major goal of this protocol is to present a fast, reliable, and scalable method to characterize protein complexes by mass spectrometry to overcome the limitations of conventional geLC-MS/MS or MudPIT protocols. This metho...
Source: Springer protocols feed by Protein Science - May 6, 2014 Category: Biochemistry Source Type: news
