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Analysis of Protein Purification

Upcoming SlideShare. Like this presentation? So for molecules treated with SDS, the pull per u nit mass in an electric field is the same, and all molecules should have the same velocity IF there is no frictional drag. But electrophoresis with SDS is always done in a gel, so that the molecules must be pulled though the pores.

INTRODUCTION

And the ease of moving through the pores depends on the diameter of the molecules. The bigger molecules are retarded or keep getting stuck and don't move as fast.

Since the molecules are all denatured into random coils, the diameter strictly depends on the length or molecular w eight. The bigger the molecular weight, the longer the coil and the slower the molecule goes. The differences in molecular weight caused by differences in the R groups are not enough to allow a separation. Gel filtration or molecular sieve chromatography. You can make a column packed with beads that have holes in them.

Methods for Protein Purification in Biotechnology

The beads are made of a gel, and the holes can be any size you want, within limits. There are many kinds of beads commercially available, but the most famous is called Sephadex.

You f ill up a column with the beads, and then fill the spaces around the beads with a buffer. Then you put your mixture of molecules on the top and wash it through the column. In this situation, immunoprecipitation cannot be carried out. An antibody successfully applied in Western blot is not necessarily applicable in IP. Adding the appropriate antibody to the proteins immobilized on the membrane we can detect the bound antibody, in case the sample contains the desired protein Figure 5.

Proteins are in denatured state on the membrane, thus the proper epitopes are accessible for the antibodies. A major drawback of the method is that protein detection is only possible in the presence of antibodies. Spots or bands containing the proteins separated by gel electrophoresis or Western blot are excised and analyzed by mass spectrometry in order to identify them Figure 5.

Protein quantitation is the determination of the absolute and relative amount of proteins in the sample. Quantitation can be carried out by gel-based or mass spectrometric methods or by their combination. The gel-based method compares two-dimensional gels to each other Figure 5.

Since for the proper comparison, a large number of technical parallels are needed, thus the introduction of the so-called fluorescence difference gel electrophoresis DIGE offers an alternative solution. The essence of this method is that one of the samples to be compared is labeled by one type of fluorescent dye and the other one is labeled by another fluorescent dye. After mixing them, the samples are run in the same gel avoiding the need of technical parallels.

The analysis of quantitative and qualitative differences of protein expression using two dimensional gel electrophoresis 2DE.

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Isolation, separation and purification

When the gel is ready it is scanned at different wavelengths by a scanner suitable for fluorescent stain detection and by the superposition of the images, the differences can easily be detected Figure 5. The analysis of quantitative and qualitative differences using difference gel electrophoresis DIGE. There are various mass spectrometry based methods:. In the course of labeling, some of the cells are cultured in a medium where some of the essential amino acids are replaced by stable isotope bearing ones. Its disadvantage is that it is expensive and can only be applied in case of cell cultures.

In iTRAQ isobaric tag for relative and absolute quantitation a labeling tag is applied, which primarily binds to the N-terminus and to the lysine amino acid residues of the proteins. Samples labeled with the different iTRAQ reagents are mixed; peaks characterizing the samples can be detected at the same m. MRM multiple reaction monitoring or SRM selected reaction monitoring is the special scan mode on triple quadrupoles.

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Quadrupoles are set in the way that the first one lets the parent ion go through only, the third quadrupole is permeable only for the proper fragment ion and the second one functions as a collision cell Figure 5. The area under the curve of the obtained signal is proportional to the concentration of the material which entered the mass spectrometer. This method can be successfully applied for the measurement of the concentration of known materials. It is widely used in pharmaceutical industry.

Detection of specific proteins using multiple reaction monitoring MRM.

Protein Biotechnology

Label-free quantitation is a purely mass spectrometric method and does not use any labeling tag.