Protein expression and purification Mammalian cells HeLa

A gross majority of classical apoptotic attributes can be quantitatively examined by flow cytometry, the preferred platform for rapid assessment of multiple cellular attributes at a single-cell level. However, sample 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 GenElute™ Mammalian Total RNA Miniprep Kit to perform RNA isolation / purification Cells - immortalized HeLa

Get tips on using Bac-to-Bac™ Baculovirus Expression System to perform Protein expression and purification Insect cells - Sf9 Drosha

Get tips on using Flp-In™ T-REx™ 293 Cell Line to perform Protein expression and purification Mammalian cells - CAL-51 BRCA1

Get tips on using Jump In™ T-REx™ HEK 293 Kit to perform Protein expression and purification Mammalian cells - HEK 293 HER2

Get tips on using pFRT to perform Protein Expression Eukaryotic cells - HeLa R19 cMBL

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.

A key signature for necrotic cells is the permeabilization of the plasma membrane. Necrosis can be quantified by several cellular and biochemical assays. When studied minutely, it reveals the difficulty in confirmation in secondary induction of necrosis in apoptotic cells. Apoptotic cells are being analyzed to shift to necrotic status owing to membrane permeability at later stages, and thus, discrimination of two cell death becomes critical. Therefore, it is crucial to use a necrosis detection kit or a defined procedure to analyze this unprogrammed form of death in response to immense chemical and physical insults.

Get tips on using pMT/BiP/V5-His A, B, & C Drosophila Expression Vectors to perform Protein expression and purification Insect cells - S2 HER2