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Engineered Human Cells: STOP HIV



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Bioluminescence
Bioluminescence is emission of light, produced by enzymatic reaction of enzyme luciferase and substrate luciferin. In molecular biology, genes expressing enzymes which catalize reactions where light is emitted are used as reporter genes. Most widely used one is firefly luciferase gene because of its big advantage; it allows us to quantitatively measure the level of gene expression. It is also a high-throughput method. The measurements are conducted directly on 96-well plates in a luminometer. After transfection and growth, cells are lysed. The substrate in appropriate cofactors are added, and the measurements are then taken automatically. Usually, cells are cotransfected with gene expresing Renilla luciferase under a constitutive promoter. This gene is used as internal standard for normalizing measurements which is necessary because of variable transfection rate.

Luciferase assay proved to be our most valuable tool due to extremely low detection limit. Therefore, despite very poor transfection rate, it was possible to detect a signal. We used this essay for successful trascription of T7 promoter genes in first stages, as well as testing our split ubiquitin and protease systems, in both cases with T7 RNA polymerase fused with transmembrane segment, and a T7 promoter – luciferase gene as a reporter.




Western blot
Western blot is a method for detection of proteins in cell lysates and determination of their molecular masses. In the first step, transfected cells grown in cell cultures are lysed and insoluble fraction is removed. Then loading buffer containing SDS and reducing agent is added to the samples. Samples are then applied to polyacrylamide gel electrophoresis which separates proteins in sample according to their molecular weight. Proteins must then be transferred to a nitrocellulose membrane by electrical current. The efficiency of transfer can be determined either by staining the gel with Coomassie brilliant blue dye or by using the pre-stained molecular weight markers which are visible on the membrane after the transfer. After the transfer, the unspecific binding sites on the membrane must be blocked in order to prevent binding the antibodies directly to membrane. Usually, milk is used for this step. Then, the nitrocellulose membrane is incubated first in solution of primary atibodies which recognise and bind to desired protein. After washing out the unbound primary antibodies, the membrane is incubated in solution of secondary antibodies that bind to the constant region of primary antibodies, and are thus species-specific. Secondary antibodies are conjugated with an enzyme horseradish peroxidase for chemiluminescence detection.

Western blot proved to be a very useful technique for determining whether proteolytic cleavage has occured. In split ubiquitin essay, the constructs CMV-CD4-CUb-GFP and CMV-CCR5-NUb were used. Using anti-GFP antibodies, proteins of different size were detected whether the GFP was cut off the transmembrane segment or remained attached to it. Similar method was used in our HIV-protease project; the construct CMV-CD4-cleavage site-GFP was used and again proteins of different size were detected when the GFP has been cleaved after cotransfection with plazmid expressing HIV protease.







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