生物化学

Apolipoprotein B–containing lipoproteins (ApoB-LPs) transport lipids throughout the circulation and are closely associated with cardiovascular disease in humans. Many aspects of ApoB-LP biology remain elusive, often due to their indirect characterization through the measurement of plasma triglycerides and cholesterol. The conventional approach provides limited information on ApoB-LPs number and size distribution, essential features that influence cardiovascular disease risk. Additionally, drug studies have historically been limited to the use of mammalian research models, which are not suited for high-throughput experiments. Therefore, we generated a reporter system (LipoGlo) utilizing a luciferase enzyme (NanoLuc) fused to the C-terminus of the zebrafish (Danio rerio) ApoBb.1 protein. In metazoans, ranging from insects to humans, each ApoB-LP contains a single ApoB molecule, such that the luminescence emitted from these transgenic fish is proportional to the total number of ApoB-LPs. The LipoGlo zebrafish reporter generates a quantitative chemiluminescent signal that can be used in plate-based assays to measure lipoprotein quantities, a gel-based assay that can measure lipoprotein size distribution, and chemiluminescent microscopy that can, for the first time, visualize lipoprotein localization in a larval zebrafish. LipoGlo, combined with the amenability of zebrafish to genetic approaches, facilitates the rapid assessment of any gene or drug’s role in ApoB-LP molecular and cell biology. This protocol describes three optimized LipoGlo assays that facilitate ApoB-LP characterization with 100× less starting material than prior assays routinely used for mammalian lipoprotein analysis.

Protein kinase B, more commonly known as Akt, is a family of three serine/threonine kinases (Akt1, Akt2, and Akt3) that play a central role in regulating processes such as proliferation, survival, metabolism, and migration through phosphorylation of downstream targets. Given its involvement in numerous cellular processes, aberrant Akt signaling is prevalent across multiple cancer types, underscoring the need for Akt kinase assays to assess activity, regulatory mechanisms, and the efficacy of targeted interventions. Most existing Akt kinase assays rely on expensive commercial kits, some of which employ pre-purified, constitutively active Akt expressed in insect cells, bypassing physiologic autoinhibition of Akt; therefore, they are not suitable for evaluating allosteric inhibitors or context-dependent regulation. Here, we describe a detailed, step-by-step protocol for a nonradioactive Akt kinase assay using epitope-tagged, recombinant Akt1 expressed in a mammalian cell line and isolated by immunoprecipitation. This method eliminates the need to co-express Akt with upstream regulatory kinases or to purify active enzyme from insect cells, a time-consuming and technically demanding process, particularly when analyzing multiple Akt mutants. Because Akt is assayed in a regulated, autoinhibited state, this protocol enables direct evaluation of allosteric inhibitors that cannot be assessed using active Akt purified from insect cells. We note, however, that Akt1 kinase activity in this assay is measured from epitope-tagged, transiently overexpressed protein, which could influence cellular signaling dynamics. Despite this limitation, the cellular context preserves key regulatory features of Akt1 autoinhibition and membrane-dependent activation that are absent in assays using purified, pre-activated kinase. Together, this protocol supports analysis of Akt kinase activity under diverse experimental conditions, including receptor stimulation, pharmacologic treatment, allosteric inhibitor exposure, and mutations, using an accessible, economical, and physiologically relevant approach.

Peroxisomal β-oxidation is a key step in jasmonic acid biosynthesis. Quantitative biochemical characterization of enzymes involved in the β-oxidation pathway is essential for validating their catalytic functions and comparing differences among genetic variants. Existing enzyme activity assays largely rely on chromatographic techniques to quantify substrate consumption or product formation, but these approaches are not well-suited for high-throughput or continuous kinetic measurements. Here, we describe a spectrophotometric assay based on a plate reader determining OsAIM1 enzymatic activity by monitoring the decrease in NADH absorbance at 340 nm. The method employs a 96-well plate reaction system, enabling real-time kinetic measurements and providing a standardized workflow for calculating reaction rates. Reaction components, protein concentration ranges, and data processing parameters were systematically optimized to ensure linearity, reproducibility, and quantitative accuracy. This assay is simple to perform, requires small reaction volumes, and offers relatively high throughput, making it suitable for functional characterization and kinetic analysis of NADH-dependent enzymes.

Transfer RNAs (tRNAs) are important regulators of translation and cellular function. Several high-throughput sequencing methods have been developed to quantitatively analyze tRNA isoacceptors in cells. However, the strong secondary structures and extensive post-transcriptional modification of most tRNA molecules present significant challenges for many reverse transcriptases, negatively impacting sequencing library preparation and causing quantification biases. Currently, the field utilizes processive next-generation reverse transcriptases (ngRTs), such as Induro (New England Biolabs) and UltraMarathonRT (RNAConnect), to address these issues. Despite being used in multiple protocols, these commercial products face little competition and remain costly. However, non-commercial alternatives, such as the original MarathonRT (MRT), are available from gene repositories. MRT is a next-generation reverse transcriptase derived from the Eubacterium rectale group II intron maturase, which can read through RNA secondary structures and chemical modifications. Here, we present a simplified expression and purification protocol for producing highly active MRT that is stable over 1 year. This cost-effective protocol yields a heterogeneous protein preparation with no discernible competing enzymatic activities; it mitigates previously reported precipitation issues, saving one day of laboratory work and eliminating two chromatography-based purification steps. Moreover, the use of the resulting protein preparation has been verified in the mim-tRNAseq pipeline, where it was shown to perform equally to the commercial alternatives Induro and UltraMarathonRT. In addition, we have developed a simple and cost-effective assay for measuring the enzymatic activity of MRT, allowing for batch comparison.

Fluorescence in situ hybridization (FISH) can be employed to study the expression and subcellular localization of nucleic acids by using labeled antisense strands that hybridize with the target RNA or DNA molecules. Likewise, immunofluorescence antibody staining (IF) takes advantage of the specific interaction between a fluorophore-labeled antibody and its corresponding antigen. This protocol reports the combination of RNA-FISH and IF antibody staining for simultaneous detection of both RNA transcripts and proteins of interest in routine formalin-fixed paraffin-embedded (FFPE) bone marrow biopsy samples. Herein, we provide a detailed description of the methodology that we have developed and optimized to study the spatial expression of two transcripts—TGFB1 and PDGFA1—in human hematopoietic (CD45⁺) and non-hematopoietic (CD271⁺) cells in the bone marrow of patients with acute lymphoblastic leukemia (ALL).

Understanding gene expression within defined neuronal populations is essential for dissecting the cellular and molecular diversity of the brain. mRNA assays provide a direct readout of gene expression, capturing transcriptional changes that may precede or occur independently of protein abundance, whereas protein assays reflect the cumulative effects of translation, modification, and degradation. Moreover, in histological analysis, immunohistochemical protein detection results in visually diffuse labeling, which makes it difficult to quantitatively assess levels and locations of expression at high resolution. Here, we present a protocol that allows for mRNA detection in single neuronal cell types with a high degree of sensitivity and anatomical resolution. This protocol combines fluorescent in situ hybridization (FISH) with immunohistochemistry (IHC) on the same tissue section. Briefly, FISH is carried out by ACDBio RNAscope^® fluorescent in situ hybridization technology, which involves processing the tissue sections, followed by signal amplification. This involves target retrieval, probe hybridization, and signal enhancement. Then, the tissue section is processed for IHC, which involves blocking nonspecific sites and incubation with primary antibodies, followed by development of a fluorescent signal with secondary antibodies. Typically, visual mRNA detection with FISH can be seen as individual puncta, whereas targeting the protein with an antibody results in filled cells or processes. The variation in staining pattern allows for the quantification of distinct mRNA transcripts within different neuronal populations, which renders co-localization analyses easy and efficient.

Evaluating RNA–protein interactions is key to understanding post-transcriptional gene regulation. Electrophoretic mobility shift assays (EMSAs) remain a widely used technique to study these interactions, revealing information about binding affinities and binding modalities, including cooperativity and complex formation. Here, we detail, in a step-by-step protocol, how to perform EMSAs. We describe how to generate, purify, and quantitate ³²P-radiolabeled RNA by in vitro transcription, as well as the expression and purification of recombinant RNA-binding proteins in E. coli using ELAV as an example. We then describe how to set up binding reactions using serial dilutions in a microtiter plate format of recombinant ELAV and in vitro–transcribed RNA and how to perform EMSAs using native low-crosslinked acrylamide gels, with detailed graphically supported instructions and troubleshooting guides.

Three-dimensional immunohistochemistry (3D-IHC) shows the organization of molecular assemblies in the context of tissue architecture. Deep and rapid antibody penetration into 3D tissues and highly sensitive detection are crucial for high-throughput analysis of 3D-IHC imaging. Here, we provide a detailed protocol for a nanobody (nAb)-based 3D-IHC technique, namely POD-nAb/FT-GO 3D-IHC, for high-speed and high-sensitivity detection of targets within 1-mm-thick mouse brain tissues. Peroxidase-fused nAb (POD-nAb) is a genetically encoded recombinant antibody, which consists of a camelid nAb and a variant of horseradish peroxidase, and fluorochromized tyramide-glucose oxidase (FT-GO) is a fluorescent tyramide signal amplification (TSA) system. POD-nAb/FT-GO 3D-IHC incorporates three main components: 1) tissue permeabilization, 2) POD-nAb binding, and 3) 3D-TSA reaction with FT-GO. POD-nAbs enhance signal penetration depth and allow for highly sensitive detection when combined with FT-GO signal amplification. By using the 3D-IHC protocol provided herein, we can visualize target molecules in mouse brain tissues of 1-mm thickness with drastic signal enhancement within three days. This protocol for POD-nAb/FT-GO 3D-IHC could facilitate structural and molecular interrogation of 3D tissues.

The activity of chloroplast ATP synthase (CF_oCF₁) is precisely regulated through a thioredoxin (Trx)-mediated dithiol/disulfide reaction in response to varying light conditions. This regulatory mechanism is further controlled by ΔpH formation across the thylakoid membrane. To better understand this complicating regulatory function of CF_oCF₁, a method is required to evaluate the extent of CF_oCF₁ reduction by Trx under controlled ΔpH conditions and to directly evaluate the redox state of CF_oCF₁. In this study, we present a simple in vitro procedure to assess the CF_oCF₁ reduction system using spinach thylakoids. The method consists of three key steps: (A) simple preparation of intact thylakoids from spinach leaves; (B) reduction of CF_oCF₁ on the thylakoid membrane using recombinant Trx under light irradiation; and (C) in situ determination of the redox state of CF_oCF₁ by labeling thiol groups with a maleimide reagent followed by protein detection using western blotting. The redox state of CF_oCF₁ was determined by mobility shifts on non-reducing SDS-PAGE. This protocol provides a refined strategy for elucidating the regulatory mechanism controlling energy conversion by CF_oCF₁ under fluctuating photosynthetic conditions.