siRNA / miRNA gene silencing Human Primary Endometrial Stromal Cells hsa-miR-542-3p

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - Caco-2

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - SH-SY5Y

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.

The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.

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 Gibco™Ham's F-12K (Kaighn's) Medium to perform Stem cell culture media Human myogenic progenitor cells

Get tips on using Leukocyte Alkaline Phosphatase Kit to perform Acid phosphatase assay cell type - human periodontal ligament cells

Get tips on using Gibco™ DMEM, high glucose to perform Stem cell culture media Human myogenic progenitor cells

Get tips on using Quant-iT™ RNA Assay Kit to perform RNA quantification Fuorimetric - human metastatic melanoma cells

Get tips on using HyClone Minimal Essential Medium (MEM) variations: Liquid to perform Stem cell culture media Human salivary gland stem cells