Protocols in Current Issue
0 Q&A 45 Views Jun 5, 2023

Cycloheximide (CHX) is a small molecule derived from Streptomyces griseus that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and used to observe intracellular protein degradation and to determine the half-life of a given protein in eukaryotes. Here, we present a complete experimental procedure of the CHX chase assay.

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0 Q&A 35 Views Jun 5, 2023

Phosphorus is an essential nutrient for plants. Green algae usually store excess P as polyphosphate (polyP) in the vacuoles. PolyP, a linear chain of three to hundreds of phosphate residues linked by phosphoanhydride bonds, is important for cell growth. Based on the previous method of polyP purification with silica gel columns (Werner et al., 2005; Canadell et al., 2016) in yeast cells, we developed a protocol to purify and determine the total P and polyP in Chlamydomonas reinhardtii by a quick, simplified, and quantitative method. We use hydrochloric acid or nitric acid to digest polyP or total P in dried cells and analyze P content using the malachite green colorimetric method. This method may be applied to other microalgae.

0 Q&A 130 Views Jun 5, 2023

Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins’ (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins.

Key features

• Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution.

• iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells.

• iCLIP facilitates the identification of sequence motifs recognized by RBPs.

• Allows quantitative analysis of genome-wide changes in protein-RNA interactions.

• Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples.

Graphical overview

0 Q&A 45 Views Jun 5, 2023

Lipid-conjugated pH sensors based on fluorophores coupled to lipids are a powerful tool for monitoring pH gradients in biological microcompartments and reconstituted membrane systems. This protocol describes the synthesis of pH sensors based on amine-reactive pHrodo esters and the amino phospholipid phosphatidylethanolamine. The major features of this sensor include efficient partitioning into membranes and strong fluorescence under acidic conditions. The protocol described here can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines.

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Protocols in Past Issues
0 Q&A 247 Views May 20, 2023

P18F3-based bi-modular fusion proteins (BMFPs), designed to re-direct pre-existing anti-Epstein-Barr virus (EBV) endogenous polyclonal antibodies towards defined target cells, demonstrated efficient biological activity in a mouse tumor model and could potentially represent a universal and versatile platform to develop novel therapeutics against a broad range of diseases. This protocol provides step-by-step instructions for expressing scFv2H7-P18F3, a BMFP targeting human CD20, in Escherichia coli (SHuffle®), and for purifying soluble proteins using a two-step process, namely immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography. This protocol can also be used for expression and purification of other BMFPs with alternative binding specificities.

0 Q&A 251 Views May 20, 2023

ATPase assays are a common tool for the characterization of purified ATPases. Here, we describe a radioactive [γ-32P]-ATP-based approach, utilizing complex formation with molybdate for phase separation of the free phosphate from non-hydrolyzed, intact ATP. The high sensitivity of this assay, compared to common assays such as the Malachite green or NADH-coupled assay, enables the examination of proteins with low ATPase activity or low purification yields. This assay can be used on purified proteins for several applications including the identification of substrates, determination of the effect of mutations on ATPase activity, and testing specific ATPase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of reconstituted ATPases.

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0 Q&A 242 Views May 20, 2023

T cells localized to the kidneys and vasculature/perivascular adipose tissue (PVAT) play an important role in hypertension and vascular injury. CD4+, CD8+, and γδ T-cell subtypes are programmed to produce interleukin (IL)-17 or interferon-γ (IFNγ), and naïve T cells can be induced to produce IL-17 via the IL-23 receptor. Importantly, both IL-17 and IFNγ have been demonstrated to contribute to hypertension. Therefore, profiling cytokine-producing T-cell subtypes in tissues relevant to hypertension provides useful information regarding immune activation. Here, we describe a protocol to obtain single-cell suspensions from the spleen, mesenteric lymph nodes, mesenteric vessels and PVAT, lungs, and kidneys, and profile IL-17A- and IFNγ-producing T cells using flow cytometry. This protocol is different from cytokine assays such as ELISA or ELISpot in that no prior cell sorting is required, and various T-cell subsets can be identified and individually assessed for cytokine production simultaneously within an individual sample. This is advantageous as sample processing is kept to a minimum, yet many tissues and T-cell subsets can be screened for cytokine production in a single experiment. In brief, single-cell suspensions are activated in vitro with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and Golgi cytokine export is inhibited with monensin. Cells are then stained for viability and extracellular marker expression. They are then fixed and permeabilized with paraformaldehyde and saponin. Finally, antibodies against IL-17 and IFNγ are incubated with the cell suspensions to report cytokine production. T-cell cytokine production and marker expression is then determined by running samples on a flow cytometer. While other groups have published methods to perform T-cell intracellular cytokine staining for flow cytometry, this protocol is the first to describe a highly reproducible method to activate, phenotype, and determine cytokine production by CD4, CD8, and γδ T cells isolated from PVAT. Additionally, this protocol can be easily modified to investigate other intracellular and extracellular markers of interest, allowing for efficient T-cell phenotyping.

0 Q&A 747 Views May 5, 2023

Western blotting is a universally used technique to identify specific proteins from a heterogeneous and complex mixture. However, there is no clear and common procedure to quantify the results obtained, resulting in variations due to the different software and protocols used in each laboratory. Here, we have developed a procedure based on the increase in chemiluminescent signal to obtain a representative value for each band to be quantified. Images were processed with ImageJ and subsequently compared using R software. The result is a linear regression model in which we use the slope of the signal increase within the combined linear range of detection to compare between samples. This approach allows to quantify and compare protein levels from different conditions in a simple and reproducible way.

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0 Q&A 440 Views Apr 20, 2023

Palmitoylation is a unique and reversible posttranslational lipid modification (PTM) that plays a critical role in many cellular events, including protein stability, activity, membrane association, and protein–protein interactions. The dynamic nature of palmitoylation dictates the efficient sorting of various retinal proteins to specific subcellular compartments. However, the underlying mechanism through which palmitoylation supports efficient protein trafficking in the retina remains unclear. Recent studies show that palmitoylation can also function as a signaling PTM, underlying epigenetic regulation and homeostasis in the retina. Efficient isolation of retinal palmitoyl proteome will pave the way to a better understanding of the role(s) for palmitoylation in visual function. The standard methods for detecting palmitoylated proteins employ 3H- or 14C-radiolabeled palmitic acid and have many limitations, including poor sensitivity. Relatively recent studies use thiopropyl Sepharose 6B resin, which offers efficient detection of palmitoylated proteome but is now discontinued from the market. Here, we describe a modified acyl resin–assisted capture (Acyl-RAC) method using agarose S3 high-capacity resin to purify palmitoylated proteins from the retina and other tissues, which is greatly compatible with downstream processing by LC-MS/MS. Unlike other palmitoylation assays, the present protocol is easy to perform and cost-effective.

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0 Q&A 335 Views Apr 20, 2023

In cells, p62/SQSTM1 undergoes liquid–liquid phase separation (LLPS) with poly-ubiquitin chains to form p62 bodies that work as a hub for various cellular events, including selective autophagy. Cytoskeleton components such as Arp2/3-derived branched actin network and motor protein myosin 1D have been shown to actively participate in the formation of phase-separated p62 bodies. Here, we describe a detailed protocol on the purification of p62 and other proteins, the assembly of the branched actin network, and the reconstitution of p62 bodies along with cytoskeletal structures in vitro. This cell-free reconstitution of p62 bodies vividly mimics the phenomenon in which low concentrations of protein in vivo rely on cytoskeleton dynamics to increase the local concentration to reach the threshold for phase separation. This protocol provides an easily implemented and typical model system to study cytoskeleton-involved protein phase separation.

0 Q&A 326 Views Apr 5, 2023

Paraquat is a cost-effective herbicide, widely used in many countries, that can induce severe oxidative stress in photosynthetic tissues. Studying plant herbicide resistance or antioxidant stress mechanisms requires determining the cellular paraquat level when plants are treated by paraquat. The traditional isotopic labeling method has the potential risk to cause problems to both human health and the environment. For radioisotope manipulation, special operation spaces and strict environmental inspection are also required. In addition, the radiolabeled paraquat is increasingly hard to buy due to the extended production cycle. Here, we describe a nonradioactive method to determine the paraquat level in a small number of Arabidopsis tissues or protoplasts, using a high resolution ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry (MS)/MS method. This method is highly selective and sensitive, and more environmentally compatible and technically feasible than the isotope detection method.

0 Q&A 293 Views Apr 5, 2023

Glycerol-3-phosphate (G3P) is a conserved precursor of glycerolipids that also plays an important role in plant defense. Its levels and/or metabolism are also associated with many human disorders including insulin resistance, diabetes, obesity, and cancer, among others. In plants, G3P accumulates upon pathogen infection and is a critical component of systemic acquired resistance, which confers broad spectrum disease resistance against secondary infections. G3P also plays an important role in root-shoot-root signaling in soybean that regulates incompatible interactions with nitrogen-fixing bacteria. Thus, accurate quantification of G3P is key to drawing a valid conclusion regarding its role in diverse processes ranging from lipid biosynthesis to defense. G3P quantification is further compounded by its rapid degradation in extracts prepared at room temperature.

Here, we describe a simplified procedure for accurate quantitative analysis of G3P from plant tissues. G3P was extracted along with the internal standard ribitol, derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by gas chromatography–coupled mass spectrometry using selective ion mode. This procedure is simple, economical, and efficient, and does not involve isotopic internal standards or multiple-step derivatizations.

0 Q&A 198 Views Apr 5, 2023

Zebrafish is an excellent model to study vertebrate neurobiology, but its synaptic components that mediate and regulate fast electrical synaptic transmission are largely unidentified. Here, we describe methods to solubilize and immunoprecipitate adult zebrafish brain homogenate under conditions to preserve electrical synapse protein complexes. The methods presented are well-suited to probe electrical synapse immunocomplexes, and potentially other brain-derived immunocomplexes, for candidate interactors from zebrafish brain.

0 Q&A 433 Views Mar 20, 2023

The envelope of Gram-negative bacteria consists of an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM). The OM and IM have different components of proteins and lipids. Separating the IM and OM is a basic biochemical procedure to further study lipids and membrane proteins in different locations. Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria. However, EDTA is often harmful to protein structure and function. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli. In this method, the cells are broken by a high-pressure microfluidizer, and the total cell membrane is collected by ultracentrifugation. The IM and OM are then separated on a sucrose gradient. Because EDTA is not used, this method is beneficial for subsequent membrane protein purification and functional study.

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