Electrophoretic Separation of Proteins Agarose gel electrophoresis is widely use

Question

Electrophoretic Separation of Proteins Agarose gel electrophoresis is widely used to separate molecules based upon charge, size and shape. It is particularly useful in separating charged biomolecules such as DNA, RNA and proteins. Agarose gel electrophoresis possesses great resolving power, yet is relatively simple and straightforward to perform. The agarose gel is made by dissolving the agarose powder (0.8 gms) into the boiling buffer solution (100 ml). This solution can be IX Tris-Acetate EDTA buffer (TAE)or IX Tris Borate EDTA (TBE) buffer solution. TAE buffer solution is preferable and recommended than TBE when looking at enzymatic applications for the DNA sample. The solution is then cooled to approximately 55°C and poured into a gel tray where it solidifies. The tray is submerged in a buffer- filled electrophoresis apparatus, which contains electrodes. Samples are prepared for electrophoresis by mixing them with components that will give the mixture density, such as glycerol or sucrose. This makes the samples denser than the electrophoresis buffer e samples can then be loaded with a micropipette or transfer pipet into wells that were created in the gel by a template during casting. The dense samples sink through the buffer and remain in the wells. A direct current power supply is connected to the electrophoresis apparatus and current is applied harged molecules in the sample enter the gel through the walls of the wells. Molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate towards the negative electrode cat, Withim a range, the higher the applied voltage, the faster the samples migrate. The buffer serves as a conductor of electricity and to control the pH. The pH is important to the charge and stability of biological molecules Agarose is a polysaccharide derivative of agar In this experiment, UltraSpec AgaroseTM is used. This resilient. The gel contains microscopic pores which act as a molecular sieve. The sieving properties pores faster than larger ones. Molecules can have the same molecular weight and charge but different material is a mixture of agarose and hydrocolloids which renders the gel to be both clear and of the gel influences the rate at which a molecule migrates. Smaller molecules move through the shapes. Molecules having a more compact shape (a sphere is more compact than a rod) can move faster through the pores. Factors such as charge, size and shape, together with buffer conditions, gel concentrations and voltage, affects the mobility of molecules in gels. Given two molecules of the same molecular weight and shape, the one with the greater amount of charge will migrate faster. In addition, different molecules can interact with agarose to varying degrees. Molecules that bind more strongly to agarose will migrate more slowly In this experiment, several different dye samples will be applied to an agarose gel electrophoresis and their rate and direction of migration will be observed. Dyes A, B, C and D are all negatively charged at neutral pHs. However, these molecules differ with respect to their structure, chemical composition and the amount of charge they carry. Dye F has a net positive charge and therefore will migrate in the opposite direction of the other dyes. This experiment will also demonstrate the ab of dyes into their individual components by the application of a combination of dyes to the same sample well.

Explanation / Answer

This is a simple gel electrophoresis.

to start take the volume to TAE based on the size of your setup,

in that, add 0.8% agarose, ex for 100 ml, add 0.8g and for 50 ml, add 0.4g and so on.

heat the gel until it is boiling and appears to be clear liquid

then allow it to cool until it reaches nearly touchable temperature (40 degrees)

pour the gel on casting tray with COMB in place or you can place the COMB immediately after casting the gel.

allow it to cool down and solidify.

remove the comb and place the gel in gel electrophoresis unit containing TAE

add your samples in each well created by comb

run the setup of gel electrophoresis

then observe under white light or gel dock.

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