Purpose Of SRP Experiments Flashcards
Cell culture
Cell culture is a process where cells are removed from the organism and introduced in to an artificial environment with favourable conditions for growth. This allows for researchers to study and learn more abt the cells
Trypan blue
Trypan blue is an azo dye. It is used as a vital stain to selectively colour dead tissues or cells blues live cells or tissues with intact cell membranes are not coloured
MTT assay
TheMTT assayis acolorimetric assayfor assessing cell metabolic activity.[1]NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insolubleformazan, which has a purple color. MTT is reduced from yellow to purple formazan. The mechanism of reduction of the dyes will also determine the amount of product
Western blotting
A western blot is a laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type. Western blots can also be used to evaluate the size of a protein of interest, and to measure the amount of protein expression. This procedure was named for its similarity to the previously invented method known as the Southern blot.
Western blotting Bradford assay
The Bradford Protein Assay is a simple spectroscopic technique which is used in laboratory research to measure the total concentration of protein in a particular sample. The principle of the procedure revolves around the concept that the maximum absorbance of acidic Coomassie Brilliant Blue G-250 alters when protein binding occurs. The anionic state of the reagent is stabilised through ionic and hydrophobic interactions, resulting in a noticeable colour change. Consequently, the Bradford Protein Assay is often a vital step of sample preparation before comprehensive SDS-PAGE and Western Blot analysis in antibody research. Factors such as loading time, cuvette material and dye temperature can all affect the results which are drawn from the process.
Western blotting SDS page
SDS-PAGE(sodium dodecyl sulfate–polyacrylamide gel electrophoresis), the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length. It usessodium dodecyl sulfate(SDS) molecules to help identify and isolateproteinmolecules.
• SDS-PAGE is adiscontinuous electrophoretic systemwhich is commonly used as a method to separateproteinswith molecular masses between 5 and 250KDa.
Western blotting semi dry transfer
Semi-dry transfer:paper > gel > membrane > paper (all wetted in transfer buffer)
Wet transfer:sponge > paper > gel > membrane > paper > sponge
In semi-dry Western Blot, the electrodes are placed directly in contact with the gel/nitrocellulose membrane sandwich to provide a fast, efficient transfer. The polyacrylamide gels must be equilibrated in transfer buffer, to remove electrophoresis buffer salts and detergents, and the nitrocellulose membranes and filter papers arepre-wetted, but that is all the buffer that is required. It is important to exclude excess moisture and air bubbles trapped in the filter papers and membrane when setting up the transfer, usually a pipet rolled over the surface will take care of this, but other than that, the set-up for this process is extremely simple.
Western blotting chemiluminescence
Usingchemiluminescenceallows multiple exposures to be made, which enables optimization of signal to noise. The detection reagents can be removed and the entireblotreprobed to visualize another protein or to optimize detection of the first protein.
PCR
Polymerase chain reaction(PCR) is a method used widely in molecular biology to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail.
DNA purification
Extract ample amounts of your genomic and/or plasmidDNAsample from a limited source to satisfy the requirements of your research.Purifyit to reduce the number of contaminants that can compromise the results of your research and shorten the shelf-life of your precious samples
Cloning DNA
DNA cloningis used to create a large number of copies of a gene or other piece ofDNA. Thecloned DNAcan be used to: Work out the function of the gene. Investigate a gene’s characteristics (size, expression, tissue distribution)
Bacterial transformation
Thepurposeof this technique is to introduce a foreign plasmid intobacteria, thebacteriathen amplifies the plasmid, making large quantities of it.
Transfection
The main purpose of transfection is to study the function of genes or gene products, by enhancing or inhibiting specific gene expression in cells, and to produce recombinantproteinsin mammalian cells
Zebra fish phalloidin stain
Fluorescent conjugatedPhalloidinis astainthat allows for visualization of F-actin. In immunohistochemistry, primary antibodies and fluorescent conjugated secondary antibodies can be used to visualize subcellular localization and relative amounts of proteins of interest.
Zebra fish in situ hybridisation
The in-situ hybridization uses a labelled complementary RNA strand to localize a specific mRNA sequence in a tissue. This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes
