protein-expression-and-purification-mammalian-cells-hek-293-mt-pa

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat pulmonary artery smooth muscle cell (pPASMC)

Get tips on using FuGENE® 6 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat pulmonary artery smooth muscle cell (pPASMC)

Get tips on using PolyMag Neo to perform DNA transfection Mammalian cells - Immortalized cell lines SH-SY5Y

Get tips on using PolyFect Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines CHO

Get tips on using EZ-10 Spin Column PCR Product Purification kit to perform DNA gel extraction / PCR product purification Product size > 15Kb

Get tips on using Cytotoxicity Detection Kit (LDH) to perform Live / Dead assay mammalian cells - RAW 264.7

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines PANC-1

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 Live and Dead Cell Assay (Abcam) to perform Live / Dead assay yeast - Syspastospora parasitica