siRNA / miRNA gene silencing Human DuCaP

TUNEL assay is the cell death detection method where the biochemical marker of apoptosis is DNA fragmentation. The assay involves the microscopical detection of generated DNA fragments with free 3'-hydroxyl residues. in apoptotic cells using enzyme terminal deoxynucleotidyl transferase (TdT) which adds biotinylated nucleotides at the site of DNA breaks. Major challenges of this method involve proper access of the enzyme which could be hampered by poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue). This issue can be resolved by optimizing the incubation time with Proteinase K or CytoninTM.

Get tips on using Naive Pan T Cell Isolation Kit, human to perform Cell Isolation Naive Pan T cell

DNA damage assay is a standard method for determining in-vivo/in-vitro genotoxicity by measuring the breaks in the DNA chain of animal and plant cells. Initial DNA damage leads to cell cycle arrest and, at the final stages, leads to induction of senescence or cell death (apoptosis, necrosis, autophagy, or mitotic catastrophe). Detection of DNA damage from mild to moderate to severe is challenging when studying genotoxicity in the pool of cells. It is favorable to use DNA damage assay kits available for prominent identification of the extent of damage in the analysis.

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Get tips on using FITC anti-human/mouse Granzyme B Antibody to perform Flow cytometry Anti-bodies Mouse - Granzyme B

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Deletion K562 c-Myb gene

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

RNA-Seq is a method to sequence RNA by applying Next Generation Sequencing (NGS). The quality of RNA is critical for the success of RNA-Seq. The integrity of RNA is measured by the RNA integrity number (RIN). RIN is computed from RNA electrophoresis and electropherogram profiles (the peak area of the 28S rRNA should be approximately twice the peak area of the 18S rRNA). If you get the RIN value lower than 7, the possibility of getting the low quality of RNA-seq data is high. To get a high quality RNA, it is better to work with fresh samples or snap-freeze the tissues in liquid nitrogen as quickly as possible and store them at -80°C until further use. Make sure designated areas and all your filter tips, microfuge tubes, plastic, and glassware are RNase-free.

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Get tips on using Proteome Profiler™ Human Apoptosis Array Kit to perform Apoptosis assay cell type - Array of apoptotic proteins