These approaches could be extended to include other signal transduction proteins and target different organelles.Clinical tissue archives represent an invaluable source of biological information. Formalin-fixed, paraffin-embedded (FFPE) tissue can be used for retrospective investigation of biomarkers of diseases and prognosis.Recently, the number of studies using proteome profiling of samples from clinical archives has markedly increased. However, the application of conventional quantitative proteomics technologies remains a challenge mainly due to the harsh fixation process resulting in protein cross-linking and protein degradation. In the present chapter, we demonstrate a protocol for label-free proteomic analysis of FFPE tissue prepared from human cardiac autopsies. The data presented here highlight the applicability and suitability of FFPE heart tissue for understanding the molecular mechanism of cardiac injury using a proteomics approach.The manufacturing and purification of therapeutic recombinant proteins expressed by cultivated mammalian cells into the cell culture medium leaves the potential for contamination by host cell proteins (HCPs). Validation and quality control testing of any therapeutic protein needs to include a test to show that HCP contamination is at a minimal level. The presence of residual mammalian HCPs during purification and in the final drug product is typically determined using enzyme linked immunosorbent assay (ELISA), which is regarded as the gold standard. The complexity and heterogeneity of HCPs, which include proteins with significant differences in molecular weight (MW), isoelectric point (pI) and hydrophobicity, poses a challenge to detection and quantitation. Two-dimensional gel electrophoresis (2-DE) is one of the most powerful technologies for studying complex protein profiles and is a valuable analytical method in biologics manufacturing. In the purification process, it is very important to know the nature and composition of HCPs, and this information can be used in a rational process design in order to minimize HCPs from the product. Additionally, 2-DE in combination with western blotting can support ELISA development and quality control for the comprehensive immunochemical detection of HCPs by estimating the recognition capacity of the polyclonal serum used in those assays. Here, we present a standardized 2-DE western blotting protocol which takes into account the latest developments in sample preparation of HCPs, protein electrophoresis, protein transfer, immunostaining, and imaging.Capture of host cell proteins (HCPs) from cell culture production harvests is critical to ensure the maximum levels specified by international regulatory bodies of product purity for therapeutic monoclonal antibodies (mAbs). Peptide ligands that selectively target the whole spectrum of the HCPs, while letting the mAb product flow through unbound, are an ideal complement to the affinity-based capture step via Protein A chromatography. In this work, we describe the development of HCP-binding peptide ligands, especially focusing on the steps of (1) peptide selection via library screening and (2) quantification of HCP removal via proteomics by mass spectrometry.The Western blot is widely used in the study of protein biochemistry, but it is notoriously labor-intensive, and it is limited in its reproducibility and quantification, among many other challenges. By contrast, capillary-based protein separation and immunodetection, known as Simple Western™, overcomes many of the challenges associated with the traditional Western blot, and it is quickly gaining traction as a replacement for traditional Western blot analysis. The advantages that capillary-based immunoassay offers include ease of use, automation, reproducibility, quantification, and even built-in total protein normalization. In this chapter, we describe protocols for the two basic types of capillary-based immunodetection assays, one by molecular weight separation and the other by charge separation. In both methods, protein samples are separated in the capillary followed seamlessly by immunodetection with chemiluminescent or fluorescent antibodies for highly sensitive and specific detection of target proteins.Western blotting continues to be a workhorse assay in laboratories throughout the world. The utility, low cost and accessibility of western blotting have allowed the technique to remain in practice, despite being developed over 40 years ago. Advances in antibody specificity, chemiluminescent formulations, properties of fluorescent molecules and imaging techniques provide gains in sensitivity, dynamic range, and ease of use. Here we discuss such aspects for the users' consideration when planning and executing western blots, to take full advantage of contemporary practices.Western blotting is one of the most used techniques in research laboratories. It is popular because it is an easy way of semiquantifying protein amounts in different samples. In Western blotting, the most commonly used method for controlling the differences in the amount of protein loaded is to independently quantify housekeeping proteins (typically actin, GAPDH or tubulin). Zilurgisertib fumarate datasheet Another less commonly used method is total protein normalization using stains, such as Ponceau S or Coomassie Brilliant Blue, which stains all the proteins on the blots. A less commonly used but powerful total protein staining technique is stain-free normalization. The stain-free technology is able to detect total protein in a large linear dynamic range and has the advantage of allowing protein detection on the gel before transblotting. This chapter discusses the theory, advantages, and method used to do total protein quantification using stain-free gels for normalization of Western blots.Over the last decade, the skeletal muscle as a secretory organ has gained importance. A growing number of peptides is described which are produced and released by the muscle fibers and work in an autocrine, paracrine, and endocrine fashion. The contraction-induced secretion of these myokines is considered to contribute to the health promoting effects of exercise. To gain further insights into the molecular processes that occur during contraction, an in vitro exercise model, electric pulse stimulation (EPS), was established. Recent publications show that this model is suitable to electrostimulate human skeletal muscle cells and thus mimic muscle contraction in vitro. Here, we provide a detailed protocol for the proteomics-based analysis of the human muscle secretome, starting with the cultivation of human myotubes and ending with sample preparation for targeted and untargeted proteome analysis of the cell culture supernatant. This workflow should allow for deeper insights into the complex nature of the muscle secretome and the identification of new myokines which might help to understand the cross talk of the working muscle with different organs and the beneficial effects of exercise.