Site Directed Mutagenesis (SDM) Human Point mutation THP-1

Get tips on using caveolin-1 Antibody (7C8): sc-53564 to perform Western blotting Caveolin-1

Get tips on using claudin-1 Antibody (XX7): sc-81796 to perform Western blotting Cclaudin-1

Get tips on using Mouse Dkk-1 Quantikine ELISA Kit to perform ELISA Mouse - Dkk-1

Reporter gene assays are designed to test the regulation of the expression of a gene of interest. This is usually done by linking the promoter of the gene of interest with a gene such as a firefly luciferase, which can be easily detected by addition of luciferin that leads to an enzymatic reaction to produce luminescence. The enzymatic reaction can be correlated to the expression of the gene of interest. Another luciferase gene that can be used is Renilla luciferase. For an appropriate luciferase assay: 1. the reporter should express uniformly in all cells, 2. specifically respond to effectors that the assay intends to monitor, 3. have low intrinsic stability to quickly reflect transcriptional dynamics. It is important to have an equal number of cells plated in each testing condition to avoid any incorrect readouts. Reporter assays could be single or dual reporter assays. The reporter could be both luciferases. Most dual-luciferase assays involve adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction. Dual-luciferase assays have some advantages, including 1. reduces variability, 2. reduces background, 3. normalizes differences in transfection efficiencies between samples.

Get tips on using LAMP-1 Antibody (H4A3) to perform Autophagy assay cell type - Proximal tubular cells (rPT)

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

Get tips on using PE Mouse Anti-Human CD31 to perform Flow cytometry Anti-bodies Human - CD31/PECAM-1

Get tips on using MrNV-pGEX-6P-1 to perform Protein Expression Prokaryotic cells - E. coli MrNV capsid

Get tips on using pIRES2-EGFP-PBD-1 to perform Protein Expression Prokaryotic cells - E. coli PBD1-EGFP

Get tips on using pGEX-6P-1 Vector to perform Protein expression and purification Bacteria - Escherichia coli FleN