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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.2-D Western Blotting for Evaluation of Antibodies Developed for Detection of Host Cell Protein
Recombinant proteins generated for therapeutic use must be substantially free of residual host cell protein (HCP). The presence of host cell protein (HCP) is usually assayed by ELISA using a polyclonal antibody mixture raised against a population of proteins derived from the host cell background. This antibody should recognize as high a proportion as possible of the potential HCPs in a given sample. A recommended method for evaluating the assay involves two-dimensional electrophoretic separation followed by Western blotting. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Stain-Free Technology
Western blotting is a commonly used laboratory technique for semi-quantifying protein amounts. It is important when quantifying protein expression to account for differences in the amount of total protein loaded onto the gel using a loading control. Common loading controls include housekeeping proteins, such as β-actin or GAPDH, quantified by Western blot, or total protein, quantified using a stain such as Coomassie Brilliant Blue or Ponceau S. A more recently developed method for total protein quantification utilizes stain-free technology, which has a linear dynamic detection range and allows for protein detection on...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
In-Gel Peptide IEF Sample Preparation for LC/MS Analysis
The technique of proteolytically digesting a sample and identifying its protein components by liquid chromatography followed by mass spectrometry (LC-MS) is a widely used analytical tool. Prior fractionation by isoelectric focusing (IEF) may be performed to increase the depth of proteome coverage. Here, we describe a method for in-gel IEF separation of a proteolytic digest that utilizes commercially available immobilized pH gradient (IPG) strips and a widely used IEF instrument. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Enrichment and Identification of Bacterial Glycopeptides by Mass Spectrometry
Large-scale analysis of protein N- and O-linked glycosylation by mass spectrometry has traditionally been performed in eukaryotes by parallel approaches aimed at elucidating glycan structures (glycomics) and their formerly glycosylated peptides (glycoproteomics) without reference to their intact state. Such analyses depend heavily on commercial glycosidases (e.g. protein N-glycosidase F) that can remove glycans from the peptide backbone for separate analyses. Bacterial glycosylation has only recently been identified as a widespread phenomenon. In many cases however, unique bacterial sugars preclude enzymatic removal, there...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Neutral Phosphate-Affinity SDS-PAGE System for Profiling of Protein Phosphorylation
In this chapter, we describe a standard protocol for phosphate-affinity SDS-PAGE that uses a dizinc(II) complex of the phosphate-binding molecule Phos-tag in conjunction with a neutral-pH gel system (Zn2+–Phos-tag SDS-PAGE) to detect shifts in the mobilities of phosphoproteins. A previous protocol for affinity electrophoresis that uses polyacrylamide-bound Mn2+-Phos-tag and Laemmli’s buffer system under conditions of alkaline pH has limitations in separating certain phosphoproteins. The current protocol provides major improvements in separation and detection of various phosphorylated protein species. We here in...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Principles and Examples of Gel-Based Approaches for Phosphoprotein Analysis
Methods for analyzing the phosphorylation status of proteins are essential to investigate in detail key cellular processes, including signal transduction and cell metabolism. The transience of this post-translational modification and the generally low abundance of phosphoproteins require specific enrichment and/or detection steps prior to analysis. Here, we describe three gel-based approaches for the analysis of differentially expressed phosphoproteins. These approaches comprise (1) the sequential fluorescence staining of two-dimensional (2-D) gels using Pro-Q® Diamond and SYPRO® Ruby dyes to visualize and quantify...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Protein Profiling and Phosphoprotein Analysis by Isoelectric Focusing
We present an experimental workflow for simultaneous analysis of the proteome and phosphoproteome with no additional enrichment for phosphoproteins/phosphopeptides. Our approach is based on isoelectric focusing (IEF) which allows the separation of peptide mixtures on an immobilized pH gradient (IPG) according to their isoelectric point. Due to the negative charge of the phosphogroup, most of the phosphopeptides migrate toward acidic pH values. Peptides and phosphopeptides are then identified by mass spectrometry (MS) and phosphopeptide spectra are manually checked for the assignment of phosphorylation sites. Here, we apply...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation
Post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and they are associated with a range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are generally present in substoichiometric levels and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task requiring highly specialized and sensitive enrichment methods. Currently, several...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Optimization of Cell Lysis and Protein Digestion Protocols for Protein Analysis by LC-MS/MS
Mass spectrometry-based proteomics is the method of choice for analyzing (sub-) proteomes from virtually any source; with cells grown in culture being the most frequently investigated samples. Using HeLa cells, we describe a strategy for the optimization of protocols for whole proteome analysis. We cover cell lysis, protein precipitation and digestion, comparing the results obtained using different parameters and offering various possibilities for the optimization of a proteomic workflow which can be used for virtually any type of animal cell grown in culture. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Mechanical/Physical Methods of Cell Distribution and Tissue Homogenization
This chapter covers the various methods of Mechanical Cell Disruption and Tissue Homogenization that are currently commercially available for processing minute samples (<1 mL) to larger production quantities. These mechanical methods of lysing do not introduce chemicals or enzymes to the system. However, the energies needed when using these “harsh” methods can be high and destroy the very proteins being sought. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Identification of Protein N-Termini Using TMPP or Dimethyl Labeling and Mass Spectrometry
Determination of a protein’s N-terminal sequence can be important for the characterization of protein processing. To increase the confidence of protein N-terminal identification, chemical derivatization of the N-terminal amine group by (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) or dimethyl labeling followed by mass spectrometric analysis is commonly performed. Using this approach, proteins can be separated by SDS-PAGE, and the protein N-terminus of interest is labeled by TMPP or dimethyl in-gel before tryptic digestion and LC-MS analysis. The N-terminus of a protein can th...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Step-by-Step Preparation of Proteins for Mass Spectrometric Analysis
Nowadays, identification of proteins from biological samples by mass spectrometry is widely used. In principle there are two scenarios. Proteins are pre-fractionated in some way, e.g. by gel electrophoresis or are analyzed as complex mixture (shot gun). Shot gun proteomics became recently more popular, because of technological developments on the mass spectrometer side which allows now the identification of several thousand proteins from complex biological matrix. However, in many cases it is still useful to separate proteins first in a gel. But not only mass spectrometer technology made progress. This is also true for the...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Depletion of RuBisCO Protein Using the Protamine Sulfate Precipitation Method
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a major high-abundant protein (HAP) in the plant leaves which hinders analysis of low-abundant proteins (LAP). In this chapter, we describe a highly simple RuBisCO depletion method using protamine sulfate (PS). Addition of 0.1 % PS is sufficient to precipitate the RuBisCO from the leaf extracts of diverse plants including monocots and dicots. Our results of SDS-PAGE, Western blotting, and two-dimensional gel electrophoresis showed that both large and small subunits of RuBisCO were precipitated in the pellet fractions, while LAPs were enriched in the supernatant f...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
Chloroplast Isolation and Affinity Chromatography for Enrichment of Low-Abundant Proteins in Complex Proteomes
Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an ab...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
A Protocol for Exosome Isolation and Characterization: Evaluation of Ultracentrifugation, Density-Gradient Separation, and Immunoaffinity Capture Methods
Exosomes are 40–150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reve...
Source: Springer protocols feed by Protein Science - March 30, 2015 Category: Biochemistry Source Type: news
