Protein expression and purification Yeast Pichia pastoris

Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

Get tips on using Yeast Extract to perform Bacterial cell culture media Legionella pneumophilia

Get tips on using Flp-In™ T-REx™ 293 Cell Line to perform Protein expression and purification Mammalian cells - HeLa ChaC1

The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.

Get tips on using Cytoplasmic and Nuclear RNA Purification Kit to perform RNA isolation / purification Cells - immortalized Akata

Get tips on using Cytoplasmic and Nuclear RNA Purification Kit to perform RNA isolation / purification Cells - immortalized JY

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

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.