CRISPR Rat Deletion AR42J

Get tips on using Annexin V-FITC Apoptosis Detection Kit to perform Apoptosis assay cell type - RAW 264.7

Get tips on using Ras (D2C1) Rabbit mAb #8955 to perform Western blotting Ras

Get tips on using Luminescent β-galactosidase Detection Kit II to perform Reporter gene assay β-galactosidase substrates - RAW 264.7

Get tips on using ROS-ID® Total ROS/Superoxide detection kit to perform ROS assay cell type - Raw 264.7

Get tips on using In Situ Cell Death Detection Kit, TMR red to perform TUNEL assay cell type - Rabbit synovial fibroblasts

Get tips on using RIPA Buffer to perform Protein isolation Mammalian cells - Rat_Liver

Get tips on using RIPA Buffer to perform Protein isolation Mammalian cells - Rat_Circumvallate papillae

Get tips on using RIPA Buffer (10X) to perform Protein isolation Mammalian cells - Rat_Renal tissue

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.

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.