Midkine is a secreted protein that acts as a growth factor or cytokine involved in cell survival and inflammatory processes. It accumulates in amyloid plaques, which are hallmarks of Alzheimer\'s Disease (AD). The reproducibility of Midkine research would be enhanced if the community had access to well-characterized anti-Midkine antibodies. In this study, we characterized 8 commercial Midkine antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in a knockout cell line and isogenic parental control. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Immunoprecipitation is one of the most effective methods for enrichment of lysine-acetylated peptides for comprehensive acetylome analysis using mass spectrometry. Manual acetyl peptide enrichment method using non-conjugated antibodies and agarose beads has been developed and applied in various studies. However, it is time-consuming and can introduce contaminants and variability that leads to potential sample loss and decreased sensitivity and robustness of the analysis. Here we describe a fast, automated enrichment protocol that enables reproducible and comprehensive acetylome analysis using a magnetic bead-based immunoprecipitation reagent.

Of all the chemical modifications of RNAs, the N6-methyladenosine (m6A) modification is the most prevalent and well-characterized RNA modification that is functionally implicated in a wide range of biological processes. The m6A modification occurs in hepatitis B virus (HBV) RNAs and this modification regulates the HBV life cycle in several ways. Thus, understanding the mechanisms underlying m6A modification of HBV RNAs is crucial in understanding HBV infectious process and associated pathogenesis. Here, we describe the currently utilized method in the detection and characterization of m6A-methylated RNAs during viral infection.

The nucleocapsid protein (N) in Rift Valley fever virus is an RNA-binding protein that functions in viral transcription, replication, and packaging. In this chapter, the method for studying protein-RNA interactions in context of viral infection using individual nucleotide resolution, cross-linking, immunoprecipitation, and sequencing (iCLIP-seq) is explained. The method is useful for identifying the interactions between both host and viral RNAs with N and can identify RNA motifs that interact with the protein of interest.

Rift Valley fever virus (RVFV; genus Phlebovirus, family Phenuiviridae, order Bunyavirales) is a mosquito-borne zoonotic pathogen endemic in Africa. Its negative-stranded genomic RNA (vRNA) is divided into three segments termed L, M, and S. Both vRNAs and antigenomic cRNAs are encapsidated by viral nucleoprotein (N) to form nucleocapsids, which constitute the template for genome transcription and replication. Based on a number of electron microscopy and structural studies, the viral RNAs of negative-strand RNA viruses, including phleboviruses, are commonly considered to be entirely and uniformly covered by N protein. However, high resolution data supporting this notion was missing to date.Here, we describe a method how to globally map all N-RNA interactions of RVFV by using iCLIP (individual-nucleotide resolution UV cross-linking and immunoprecipitation). The protocol is based on covalent cross-linking of direct protein-RNA interactions by UV irradiation. Following sample lysis, a selective isolation of N in complex with its RNA targets is achieved by immunoprecipitation. Then, N-RNA complexes are separated by SDS-PAGE, and after membrane transfer, RNA is isolated and subjected to library preparation and high-throughput sequencing. We explain how the standard iCLIP protocol can be adapted to RVFV N-RNA interaction studies. The protocol describes mapping of all N interactions with the vRNAs and cRNAs derived either from RVFV particles or from infected cells.

Affinity enrichment coupled with liquid chromatography-tandem mass spectrometry (AE-LC-MS/MS) enables a comprehensive study of virus-host protein-protein interactions in cells and tissues infected with Rift Valley fever virus (RVFV) or ectopically expressing RVFV proteins. Depending on the research question, different experimental setups with carefully chosen controls are needed. Here, we describe the detailed workflow of sample preparation, processing, and cleanup, while also outlining critical points to consider when designing and performing AE-LC-MS/MS experiments.

Intact-mass spectrometry has huge potential for clinical application, as it enables both quantitative and qualitative analysis of intact proteins and possibly unlocks additional pathophysiological information via, e.g., detection of specific post-translational modifications (PTMs). Such valuable and clinically useful selectivity is typically lost during conventional bottom-up mass spectrometry. We demonstrate an innovative immunoprecipitation protein enrichment assay coupled to ultrahigh performance liquid chromatography quadrupole time-of-flight high resolution mass spectrometry (UPLC-QToF-HRMS) for the fast and simple identification of the protein tumor marker Neuron Specific Enolase Gamma (NSEγ) at low endogenous concentrations in human serum. Additionally, using the combination of immunoaffinity purification with intact mass spectrometry, the presence of NSEγ in an acetylated form in human serum was detected. This highlights the unique potential of immunoaffinity intact mass spectrometry in clinical diagnostics.

Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and post-translational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MN) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including polymerase chain reaction (PCR), quantitative PCR (qPCR), or sequencing. This protocol outlines each of these steps for both yeast and human cells. This chapter includes a contextual discussion of the combination of ChIP with DNA analysis methods such as ChIP-on-Chip, ChIP-qPCR, and ChIP-Seq, recent updates on ChIP-Seq data analysis pipelines, complementary methods for identification of binding sites of DNA binding proteins, and additional protocol information about ChIP-qPCR and ChIP-Seq.

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Lysosomes constitute the main degradative compartment of most mammalian cells and are involved in various cellular functions. Most of them are catalyzed by lysosomal proteins, which typically are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance and to enable profiling of lysosomal content, lysosomes are often enriched. Two approaches have gained popularity in recent years, namely, superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). The effect of these approaches on the lysosomal proteome has not been investigated to date. We addressed this topic through a combination of both techniques and proteomic analysis of lysosome-enriched fractions. For SPIONs treatment, we identified altered cellular iron homeostasis and moderate changes of the lysosomal proteome. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially for lysosomal membrane proteins and those involved in protein trafficking. Furthermore, we established a combined strategy based on the sequential enrichment of lysosomes with SPIONs and TMEM-IP. This enabled increased purity of lysosome-enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow-through and eluate fractions, additional insights into the properties of individual approaches. All data are available via ProteomeXchange with PXD048696.