siRNA / RNAi /miRNA transfection Human Cells HESC

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

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

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - STTG1

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

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

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.

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

Acid phosphatase detection heavily relies on determining the concentration of tartrate-resistant acid phosphatase (TRAP) in the sample. Hence, sample preparation is very crucial and it should be done strictly as per kit manufacturer instructions to avoid any inconsistency and poor sensitivity.

Cell cytotoxicity assays measure the ability of certain compounds or chemical mediators to reduce the viability of the cells. The term cell cytotoxicity assay can sometimes be used interchangeably with cell proliferation assay. Healthy living cells can be identified by the use of formazan dyes, protease biomarkers or by measuring ATP content. The formazan dyes are chromogenic products formed by the reduction of tetrazolium salts by dehydrogenases, such as lactate dehydrogenase (LDH) and reductases that are released during cell death. Common tetrazolium salts include INT, MTT, MTS and XTT. Cell cytotoxicity can also be measured by using the SRB and WST-1 assays. These assays can usually be used in a high-throughput fashion and can be quantitated by measuring absorbance, colorimetry or luminescence. All these assays require similar numbers of cell plating at the initiation, a time course of treatment with the cytotoxic agent and at least triplicates for each condition at every point of analysis. Cell shrinkage, plasma membrane blebbing, cell detachment, externalization of phosphatidylserine, nuclear condensation and ultimately DNA fragmentation are well-described features of apoptosis. The assays that rely on cell membrane integrity for their function, may not be able to quantify early apoptosis. Therefore, in order to distinguish early apoptotic vs. late apoptotic or necrotic cells, additional flow cytometry techniques can be used. A combination of Annexin V and PI (propidium iodide) can be used to distinguish early (Annexin V+/PI-) and late apoptotic (Annexin V+/PI+) cells. Sometimes, caspase assays are used in order to differentiate the stages of apoptosis.