Immunohistochemistry Anti-Glial Fibrillary Acidic Protein (GFAP)

Get tips on using CelLytic™ Y Plus Kit to perform Protein isolation Yeast - Candida boidinii

Get tips on using CelLytic™ Y Plus Kit to perform Protein isolation Yeast - Saccharomyces cerevisiae

Get tips on using CelLytic™ Y Plus Kit to perform Protein isolation Yeast - Pichia pastoris

Get tips on using CelLytic™ B Plus Kit to perform Protein isolation Bacteria - Escherichia coli

Get tips on using CelLytic™ B Cell Lysis Reagent to perform Protein isolation Bacteria - Anabaena

Get tips on using CelLytic™ B Cell Lysis Reagent to perform Protein isolation Bacteria - Synechocystis

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 Ni-NTA Magnetic Agarose Beads (6 x 1 ml) to perform Protein tag Purification of His-tagged proteins

Generally it has been difficult to isolate high-quality RNA from yeast because of problems disrupting the cells. Use of enzymes to disrupt cell wall can alter gene expression profiles. Therefore, physical disruption can result in high quality RNA for all downstream processing. Use of DNAse and proteinase K will remove traces of DNA contamination and proteins respectively.

Generally it has been difficult to isolate high-quality RNA from yeast because of problems disrupting the cells. Use of enzymes to disrupt cell wall can alter gene expression profiles. Therefore, physical disruption can result in high quality RNA for all downstream processing. Use of DNAse and proteinase K will remove traces of DNA contamination and proteins respectively.