Electrophoresis Animation

Gel electrophoresis is a technique used for the separation of DNA, RNA, or protein molecules using electric current applied to gel matrix.It is used has as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting


Gel electrophoresis is performed in silica gel which inert porus medium. In electrophoresis macromolecules like DNA, RNA and protein migrate when electric current is passed through the medium. Separation of molecules depends upon two forces namely mass and charge, When the macromolecules are mixed with a buffer and applied to a gel, the electric current from one eletrode refuses the molecule while the other one attracts the molecule, this frictional force separates the molecule by size, During the process of electrophoresis molecules are forced to move through the pores when a electrical current is applied, the molecules speed depends on the strength of the field, their shape, size and strength and temperature of the buffer,separeted molecules can be seen in bands



After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.

If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.


Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.













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Column Chromatography

Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms.

The classical preparative chromatography column is a glass tube with a diameter from 5 to 50 mm and a height of 50 cm to 1 m with a tap at the bottom. A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles. A solution of the organic material is pipetted on top of the stationary phase. This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluant. Eluant is slowly passed through the column to advance the organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating funnel is put on top of the column.








Steps involved:




During chromatography support or matrix is placed in the column,the support is equilibrated with the buffer which is generally identical to buffer in which the sample is dissolved
and sample is applied to the column,in most chromotograhy protocols the protein of interest along with the protein impurities in the sample find to the matrix,the impuritis are washed away by the buffer ,which usually the same buffer which is used in equilibration,then sample is then eluded and collected by washed with appropriate buffer,if teh single buffer is used the process is reffered to as isocratic separation,if multiple buffers are used its is called has gradient,the support is then washed by regeneration buffer that prepares th column for further use and storage with exception of gel filteration ,this basic protocal is used.