Protein Expression Eukaryotic cells lettuce leaves

Get tips on using T-PER™ Tissue Protein Extraction Reagent to perform Protein isolation Mammalian cells - Rat_Renal tissue

Get tips on using T-PER™ Tissue Protein Extraction Reagent to perform Protein isolation Mammalian cells - Rat_Mesenteric fat

Get tips on using M-PER™ Mammalian Protein Extraction Reagent to perform Protein isolation Mammalian cells - SH-SY5Y

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Mammalian cells - Mouse macrophages

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Mammalian cells - Neuro 2a

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Mammalian cells - OUMS-27

Get tips on using M-PER™ Mammalian Protein Extraction Reagent to perform Protein isolation Mammalian cells - HLE-B3

Get tips on using AllPrep RNA/Protein Kit to perform RNA isolation / purification Cells - primary human epidermal keratinocytes

Protein ladders are a set of standards known as molecular weight proteins that are utilized to identify the approximate size of a protein molecule run on a PAGE gel electrophoresis. The challenges in running the ladders are the choice of appropriate protein standard as it is used as visual evidence of protein migration, transfer efficiency, and positive control. Suitable protein markers can be selected on the basis of required properties and applications, i.e., fluorescent ladder, IEF, 2D SDS-PAGE ladder, natural ladder with an isoelectric point, and optimized ladders for Western Blot chemiluminescence detection. The key factors for running a distinct protein ladder are buffer conditions, charge/voltage at migration time, and the gel's concentration.

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 has 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 the efficacy of transduction and shRNA on its target site.