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| - Fridrich and Pertus methods are quite common, as well as the Roux method. Some other methods are Heise, Zbroski-Brucem, and Vandebergh-Harris.
- Nekaj besed uvoda... 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
- A number of people have asked for an overview of what quantitative and formal methods are available for political science research. Here are a list of methods: This page could use a brief summary overview, that ties together these links. A short list:
- 2.3.3 Method declarations A method declares an executable port that can be invoked, passing a fixed number of values as arguments. MethodDeclaration: MethodHeader MethodBody MethodHeader: MethodModifiers (opt) ResultType MethodDeclarator Throws (opt) where : ResultType: Type Void MethodDeclarator: Identifier (formal parameter list optional) FormalParameter: Final (opt) Type VariableDeclaratorID
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| - Fridrich and Pertus methods are quite common, as well as the Roux method. Some other methods are Heise, Zbroski-Brucem, and Vandebergh-Harris.
- A number of people have asked for an overview of what quantitative and formal methods are available for political science research. Here are a list of methods: This page could use a brief summary overview, that ties together these links. A short list:
* Statistical Methods
* Regression Analysis
* Maximum Likelihood Estimation
* Structural Equation Modeling
* Deductive Methods
* Game Theory
* Spatial Voting Theory
* Axiomatic Theory
* Simulation Methods
* Agent-based Modeling
* Discrete Event Simulation
* System Dynamics Modeling
* Monte Carlo Simulation (common to many methods)
- Nekaj besed uvoda... 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. Previous Home Next
- 2.3.3 Method declarations A method declares an executable port that can be invoked, passing a fixed number of values as arguments. MethodDeclaration: MethodHeader MethodBody MethodHeader: MethodModifiers (opt) ResultType MethodDeclarator Throws (opt) where : ResultType: Type Void MethodDeclarator: Identifier (formal parameter list optional) FormalParameter: Final (opt) Type VariableDeclaratorID There is a compile-time error : If the body of a class have two methods with the same signature. If two formal parameters of the same method are declared to have the same name, then a compile-time error occurs. If a method parameter that is declared final is assigned to within the body of the method. A constructor is also treated as a method. Parameter names may not be re-declared as local variables of the method, or as exception parameters of catch clauses in a try statement of the method. 2.3.3.2 Method modifiers Method modifiers are one of the following; if two or more method modifiers appear in a method declaration, it is customary, though not required, that they appear in the order consistent with that shown below: Public Protected Private Abstract Static Final Synchronized A compile-time error occurs : If the same modifier occurs more than once in the method declaration If a method declaration has more than one of the access modifiers : public, protected or private. If a method declaration contains the keyword abstract and also contains any of the keywords, private, static, final, native or synchronized. An abstract method declaration introduces the method as a member, providing its signature, return type and throws clause (if any), but does not provide an implementation. A declaration of a abstract method must appear directly within an abstract class, failing which a compile-time error results. An abstract class can override an abstract method by providing another method declaration. A method that is declared static is always invoked without reference to a particular object. An attempt to reference the current object using the keyword this or the keyword super in the body of a static method results in a compile-time error. A static method cannot be declared abstract. A non-static method can always refer to the current object using the keywords this and super during the execution of the method body. A method can be declared final to prevent subclasses from overriding or hiding it. A private method and all methods declared in a final class is implicitly final as it is impossible to override them. “A final method cannot be declared abstract. “ A throws clause is used to declare any checked exceptions that can result from the execution of a method. A compile-time error occurs if any class type mentioned in a throws clause is not the class throwable or a subclass throwable. A method body is either a block of code that implements a method or simply a semi-colon indicating the lack of implementation such as in cases abstract and native methods. If a method is declared void, then its body must not contain any return statement that has a expression. If a method is declared to have return type, then every return statement in its body must have an expression. A class inherits from its direct super class all the non-private methods (whether abstract or not) of the super class that are accessible to code in the class and are neither overridden nor hidden by a declaration in the class. 2.3.3.3 Inheritance, Overriding, and Hiding A class C inherits from its direct superclass and direct superinterfaces all non-private methods (whether abstract or not) of the superclass and superinterfaces that are public, protected or declared with default access in the same package as C and are neither overridden nor hidden by a declaration in the class. An instance method m1 declared in a class C overrides another instance method, m2, declared in class A iff all of the following are true: C is a subclass of A. The signature of m1 is a subsignature of the signature of m2. Either m2 is public, protected or declared with default access in the same package as C, or m1 overrides a method m3, m3 distinct from m1, m3 distinct from m2, such that m3 overrides m2. Moreover, if m1 is not abstract, then m1 is said to implement any and all declarations of abstract methods that it overrides. A compile-time error occurs if an instance method overrides a static method. It is a compile time error if a type declaration T has a member method m1 and there exists a method m2 declared in T or a supertype of T such that all of the following conditions hold: m1 and m2 have the same name. m2 is accessible from T. The signature of m1 is not a subsignature of the signature of m2. m1 or some method m1 overrides (directly or indirectly) has the same erasure as m2 or some method m2 overrides (directly or indirectly).
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