This method is for the immunoprecipitation of Flag-Tagged RNA binding proteins from mammalian cell lines and isolation of the bound RNAs for analysis by quantitative real-time PCR. The RNA binding protein of interest should be tagged with the M2 Flag-tag and expressed in the mammalian cell line of interest (Knuckles et al., 2012). However, specific antibodies for the protein of interest can be used in conjunction with Sepharose G-beads.

Lung clearance assay tests the ability of innate immune cells (mainly NK cells) to eradicate in vivo cells injected via the tail vein of the mice. This assay helps to elucidate the role played by NK cells and their receptors (if the mice are genetically modified) against various human and mouse targets in an in vivo setting (Stern-Ginossar et al., 2008; Halfteck et al., 2009; Tsukerman et al., 2012).

MicroRNAs (miRNAs) are small non-coding RNAs of 21-24 nucleotides in length that modulate gene expression by targeting the untranslated region (UTR) of mRNA. This protocol is to be used to test the binding and activity of miRNA on putative UTR target sequences. It is based on the expression of Luciferase as a reporter gene fused to the UTR sequence in the presence of plasmids containing pre-miRNA of interest or synthetic miRNA to test in an in vitro cell culture assay.

This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides “three-dimensional” views of the stained components in order to determine the localization of a protein of interest in the tissue in its natural state and environment.

Leaf metabolism produces hydrogen peroxide (H2O2) at high rates, high level H2O2 accumulation can cause oxidative stress. This protocol describes a method for determining H2O2 concentration in tobacco leaves. In this method all extractions were performed with HClO4, neutralized, and pretreated with ascorbate oxidase to eliminate ascorbate interferences. H2O2 content was determined using a colorimetric assay spiked with an internal control. Interfering peroxides were determined in parallel using a negative control treated with catalase and subsequently subtracted.

The method of immunoelectron microscopy is intended for localization of proteins inside the cells of Chlamydomonas reinhardtii or other microalgae and cyanobacteria. This protocol was used to study localization of carbonic anhydrase Cah3 with antibodies raised in rabbit, though it can be used to localize any other abundant protein. Primary rabbit antibodies are recommended because they react quickly and specifically with proteins of C. reinhardtii. If primary antibodies other than rabbit are used, the blocking procedure and time of incubation with primary and secondary antibodies should be adjusted.

TL measurement is a useful tool for studying charge stabilization and subsequent recombination in photosystem II (PSII) in higher plants and cyanobacteria. Recombination of positive charges stored in the S2 and S3 oxidation states of the water oxidizing complex with electrons stabilized on the reduced QA and QB acceptors of PSII results in characteristic TL emissions. The TL intensity reflects the amount of recombining charges and the peak temperature is indicative of the energetic stabilization of the separated charge pair: the higher the peak temperature, the greater the stabilization. Illumination of single-turnover flash with the plant or cyanobacterial sample after a short dark adaptation induces a major TL band, called the B band which appears around at 30 °C and arises from S2/S3QB- recombination. If electron transfer between QA and QB is blocked by DCMU, the B band is replaced by the so-called Q-band arising from S2QA- recombination at around 10 °C. Illumination with a series of single-turnover flashes result in B bands oscillating with a period of 4, with a maximum at the second flash. Here we mainly described the measurements of TL B-band (charge reccombination of S2/S3QB-), Q-band (charge reccombination of S2QA-,) and period-four oscillation of the intensity of the B-band in tobacco leaves.