DNA methylation profiling Gene specific profiling UMR-106

The most widely used method for protein quantification is by spectrophotometry. The concentration of the protein in the samples is measured at an absorbance of 280 nm. The absorbance of the sample protein is then plotted against a standard curve. This method allows for total protein quantification in a sample (cell and tissue extracts). Before analysing the concentration of protein in the sample, it is important to choose the right test method.  For high protein concentration samples (above 5 - 160 mg/ml) the best method is to use the Biuret test. For low concentrations samples (between 1 - 2000µg/ml) the best methods are Lowry assay, BCA assay, Bradford assay and coomassie blue (for exact sensitivity of the test kits you use, refer to manufacturer's protocol). If the samples contain detergents like Triton X-100 then BCA assay is the best choice. For samples that have proteins larger than 3 KDa in size Bradford assay is the best choice. Each method has advantages and disadvantages, plan your analysis considering your sample characteristics.

The RNA interference (RNAi) is used to inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Two types of RNA molecules such as microRNA (miRNA) and small interfering RNA (siRNA) play a central role in RNAi. Few points have to considered to increase the transfection efficiency of siRNA. Always use healthy, actively dividing cells to maximize transfection efficiency. The confluency of cells should be between 50-70%. Always use the most appropriate siRNA concentration to avoid off-target effects and unwanted toxic side effects. Positive and negative controls should be used for each and every experiment to determine transfection efficiency.

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

Get tips on using LIVE/DEAD™ Yeast Viability Kit to perform Live / Dead assay yeast - Urediniospore

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been greatly used for studies on embryonic development and cell differentiation.iPSCs provide a stable source for either self-renewal or differentiation into suitable cells when cultured in a particular environment. Pluripotent cell culture was originally started by deriving cells from inner cell mass (ICM) from pre-implanted blastocysts, these were called embryonic stem cells. These cells after isolation can be grown on traditional extracellular matrices (like mouse embryonic fibroblasts, MEFs) or feeder-free culture systems. DMEM/F12 has been the most commonly used basal media in the culture of pluripotent cells. These cells are cultured at normal atmospheric oxygen levels, 21%, however, some studies have proposed that 4% oxygen tension may be better for hESC growth. Higher D-glucose concentration (4.2g/l) and osmolarity (320mOsm) that mimics the natural environment of embryonic tissue are optimal for the growth of hESCs. Supplements like N2 and/or B-27, in the presence of growth factors like bFGF, have been shown to increase pluripotency of these cells. bFGF, FGF2 and other ligands of receptor tyrosine kinases like IGF are also required or maintain self-renewal ability of these cells. TGF𝛃1, by its activation of SMAD2/3 signalling, also represses differentiation of iPSCs. Other compounds like ROCK inhibitors reduce blebbing and apoptosis in these cells to maintain their clonogenicity. However, an inhibitor for LIF (leukaemia inhibitory factor, which is one of the pluripotent genes) has an opposing effect. Therefore, it is important to understand the culture conditions and media composition that affect downstream signalling in hESCs or iPSCs that may lead to their differentiation.

Get tips on using LIVE/DEAD™ FungaLight™ Yeast Viability Kit, for flow cytometry to perform Live / Dead assay yeast - Urediniospore

Get tips on using GeneJET RNA Purification Kit to perform AAA for reviews

Get tips on using GeneJET Plasmid Miniprep Kit to perform Plasmid Isolation Piscirickettsia salmonis

Get tips on using GeneJET Plasmid Miniprep Kit to perform Plasmid Isolation Proteus mirabilis

Get tips on using GeneJET Plasmid Miniprep Kit to perform Plasmid Isolation Lactobacillus brevis