cell-cycle-assay-human-hela

Get tips on using 24-Well Cell Invasion Assays, Basement Membrane to perform Cell migration / Invasion cell type - MG-63

Get tips on using 24-Well Cell Invasion Assays, Basement Membrane to perform Cell migration / Invasion cell type - HT-29

Get tips on using Human IL-1 beta/IL-1F2 Quantikine ELISA Kit to perform ELISA Human - IL-1 beta

Get tips on using Human ICAM-1/CD54 Allele-specific Quantikine ELISA Kit to perform ELISA Human - ICAM-1/CD54

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.

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.

Get tips on using Polyclonal Rabbit Anti-Human Myeloperoxidase (Dako Omnis) to perform Immunohistochemistry Mouse - MPO

Get tips on using NLRP3/NALP3 (human) monoclonal antibody (Nalpy3-b) to perform Western blotting NLRP3

Get tips on using Purified anti-mouse/rat/human FOXP3 Antibody to perform Western blotting FOXP3

Get tips on using PE anti-human CD135 (Flt-3/Flk-2) Antibody to perform Flow cytometry Anti-bodies Human - CD135