- SALTING IN\/OUT<\/strong><\/li>
- SONICATION<\/strong><\/span><\/li>
- CENTRIFUGATION<\/strong><\/span><\/li>
- ISOELECTRIC FOCUSING<\/strong><\/span><\/li>
- SDS-PAGE<\/strong><\/span><\/li>
- NATIVE-PAGE<\/strong><\/span><\/li>
- WESTERN BLOT ELISA<\/strong><\/span><\/div><\/li><\/ol>\n\n\n\n
Salting in and out<\/strong><\/h2>\n<\/span>\n\n\n
In most of the cases, we got samples from a patient in the form of \u201cSERO\u201d-type samples or URINE samples respectively. In these samples, all the proteins are in a dissolved stage, from which we need to make an insoluble one. Regarding the blood samples, we need to centrifuge it first to separate the plasma-serum fractions. But in the case of the urine samples, we need to change the osmotic features of the solution. The basic method to make our proteins visible in a solution is to precipitate them. One method for this is the increasing concentration of salt (Figure 1).<\/p>\n\n\n\n
![\"Figure](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/03\/SALTIN-1024x321.png\")
<\/a>Figure 1. Protocol for liquid samples<\/figcaption><\/figure>\n\n\n\n The salting out method involves different salts like:<\/p>\n\n\n\n
NaCl, KCl, MgSO4<\/sub>, (NH4<\/sub>)2<\/sub>SO4<\/sub>, KSO4.<\/sub><\/p>\n\n\n\n
Salting out (salt-induced precipitation) is a purification method that utilizes the reduced solubility of proteins and DNA in a liquid solution of very high ionic strength. Because the salt concentration needed for a given protein to precipitate out of the solution will be different from protein to protein of interest, every time a specific salt concentration can be used to precipitate the target protein.<\/p>\n\n\n\n
The basic principle of the method is that salt compounds dissociate in aqueous solutions. When the salt concentration is increasing, some of the water molecules are attracted by the salt ions in the solution, which decreases the number of water molecules available to interact with the charged part of the protein (Figure 2).<\/p>\n\n\n\n
![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/03\/SALTIN2-1024x696.png\")
<\/a>Figure 2. Precipitation with increasing the salt concentration in the sample<\/figcaption><\/figure>\n\n\n\n In our sample there are hydrophobic amino acids and hydrophilic amino acids in protein molecules respectively. The hydrophobic amino acids usually form hydrophobic areas meanwhile hydrophilic amino acids interact with the molecules of solvation and give chance proteins to form hydrogen bonds with the surrounding water molecules in the solution. If the protein surface is enough hydrophilic, thus the protein can be dissolved in water.<\/p>\n\n\n\n
When salt is added to the solution, the protein and salt ions are competing to interact with the solvent molecules resulting in that there are fewer solvent molecules available for interaction with the protein molecules than originally.
The protein\u2013protein interactions consequently become stronger than the solvent\u2013solute interactions and the protein molecules associate by forming hydrophobic interactions with each other. Additionally, the negatively charged atoms from the salt begin to compete for interactions with positively charged biomolecules (proteins) presented in the liquid. Similarly, the positively charged cations will compete for interactions with the negatively charged molecules of the solvent.<\/p>\n\n\n\n
Unrelated\/unwanted proteins can be removed from the sample salting out as long as the solubility of the protein in various concentrations of salt solution is known exactly. After removing the precipitate by filtration or centrifugation, the desired protein can be precipitated by altering the salt concentration to the level at which the desired protein becomes insoluble.<\/p>\n<\/span> - SONICATION<\/strong><\/span><\/li>
![\"Figure](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/03\/SALTIN.png\")
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