Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.
Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.
The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.
The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.
RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. 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.
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
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
which is a good monoclonal anibody for this protein?
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