this video is designed to help you understand the basic techniques of protein purification and provides advice on how to start with your own protein purification project [Music] there are many techniques involved in the purification of proteins in this video we will discuss some of the most common ones which are salting out dialysis electrophoresis affinity chromatography iron exchange chromatography hydrophobic interaction chromatography and size exclusion chromatography but first let's clarify what protein purification is protein purification aims to isolate a single type of protein called the target protein from a biological tissue or culture the purified protein should be free of contaminants but also of the presence of various isoforms the purification of proteins finds many applications it can be used to produce proteins for biopharmaceuticals food supplements or in the detergent industry in addition it is often employed to analyze the characteristics structures and functions of proteins during scientific research before you can purify your target protein there is one question you should ask yourself which chemical structural and functional properties does the target protein have protein purification techniques are based on the exploitation of differences in the properties of the target protein and other proteins in the crude mixture basically we are looking for a characteristic that sets your target protein apart from the rest these unique features might be found in size and shape charge npi solubility or precipitation density stability hydrophobicity or ligand-binding affinities once you're clear on this you can begin the protein purification process separate your protein from a biological tissue or culture the protein purification process often begins with the destruction of the cells this can be done through chemical methods such as osmotic shock or mechanical methods such as ultrasonification afterwards larger fragments such as cell debris can be separated from the lysid by centrifugation in order to purify a protein you'll need to make sure that it's soluble unstable inside a solution which makes the choice of buffer an important one now let's start talking about the various protein purification techniques there are many protein purification techniques and often multiple are combined although each protein purification step usually results in higher purity and quality it can also lead to some degree of product loss and to higher production costs therefore an ideal protein purification strategy is one in which the highest level of purity is achieved in the fewest steps generally speaking protein purification for academic or pharmaceutical purposes requires the highest amount of purity whereas the food and detergent industries might not require protein in an ultra pure form precipitation methods these techniques separate proteins for their solubility their aim is to change the conditions of a solution in which the proteins are dissolved to such an extent that it causes the proteins to precipitate one example of these techniques is salting out salting out is based on the idea of decreasing the solubility of a protein dissolved in water by adding salt such as aluminium sulfate proteins differ in their solubility in water based on their amount of hydrophilic and hydrophobic amino acids in a solution without salt water molecules surround the hydrophilic part of the target protein and stabilize it through a hydration shell adding salt to your protein solution decreases the ratio of available hydration shell because the hydrophilic parts of the protein and the sold ions compete to interact with it when enough salt is added to the solution the protein protein interactions become stronger than the interactions between the hydrophilic parts and the hydration shell to become more stable the protein molecules associate through hydrophobic interactions the precipitated protein can then be removed from the target protein solution salting out is a low-cost environmental friendly technique and does not cause irreversible protein denaturation however its ability to purify proteins is crude and therefore it is often combined with other protein purification methods chromatography chromatography is the most commonly used purification method it allows the resolution of complex crude mixtures with very similar molecular properties however downsides to chromatography are that is often more expensive and time consuming than non-chromatographic methods this makes it more commonly used for smaller samples overall chromatography can be described as a process in which proteins in a solution called the liquid phase are separated based on differences in their chemical or physical interactions with a stationary material called the solid phase the aim is to exploit the target protein's unique interaction with the solid phase while washing away all other molecules whose interactions differ and eventually elude the target protein from the solution affinity chromatography affinity chromatography purifies proteins due to their affinity to certain ligands hereby naturally occurring affinities are more commonly added affinity tags are used for purification the most prominent affinity tag is the his-tag the his-tag has an affinity towards metal such as nickel two plus coordinated by chelators to purify a his-tagged protein the protein solution is added to chromatography beads which are complexed with a ligand such as nickel nta the tag then binds to the iron on the bead and everything else that is not bound is washed out afterwards the tagged protein is eluted from the beets with a ligand that has an even higher affinity in the previous example a high concentration of imidazole would be used to elude a his tucked protein from a nickel nta column among the chromatography techniques affinity chromatography plays a major role because it is highly specific and effective if you would like to see how affinity chromatography can be applied inside a laboratory check out our video linked above ion exchange chromatography ion exchange chromatography is based on the charge of a protein at a certain ph in case the beats of a stationary phase are positively charged negatively charged proteins cling to it this is called an ion exchange while the opposite situation in which we have a negatively charged stationary phase binding to positively charged proteins is called cation exchange after washing the column the protein is eluted with higher charged ligands for example to elude the target protein from a cat ion exchange chromatography column a high concentration of sodium ions can be used hydrophobic interaction chromatography hydrophobic interaction chromatography takes advantage of the protein's hydrophobicity remember the technique of salting out in which a high soul concentration leads to the withdrawal of the hydration shell and enables the hydrophobic regions of the protein to associate well this is exploited here a high salt concentration is added to the protein solution in which the target protein is only just soluble when the solution is added to the hydrophobic stationary phase the target protein is encouraged to bind to it because a hydrophobic interaction is more accessible than interacting with the hydration shell to elude the target protein one can continuously decrease the soul concentration which reconstitutes the hydration shell of the target protein weakening the hydrophobic interaction alternatively a change in ph could be preferred in case the changing increases the protein's net charge size exclusion chromatography science exclusion chromatography separates proteins based on their size it is different to other chromatography methods because it does not aim to bind the target protein to the beads of a stationary phase the aim of size exclusion chromatography is to sort proteins in a solution by their size this technique uses chromatography beads containing many similar sized pores when the solution is run through the column large proteins are able to migrate past the beads the quickest as they are unable to enter any of the pores small proteins on the other hand diffuse into all the pores and need to wind their way through the inside of the beads this leads to a slower migration and thus they will be eluted last size exclusion chromatography provides a simple way to remove all aggregated target protein complexes because they would be of a larger size furthermore it provides the opportunity to purify only one oligomerization state of the target protein filtration methods these methods rely on a membrane to filter the protein solution one example of these methods is dialysis during dialysis the protein solution is placed into a semi-permeable tube the filled tube is then placed into a buffer water and small ions are now able to move through the semi-permeable tube and spread out into the space larger molecules such as proteins and dna on the other hand are unable to pass through and others help back dialysis does not separate proteins it reduces the concentration of salts reducing agents or dies inside the solution or it can be used to slowly change the buffer's ph value therefore it is rarely applied on its own to purify proteins electrophoresis electrophoresis sorts proteins by their charge size and shape a protein's charge depends on the environmental ph the isoelectric point of a protein in short pi is the ph at which the protein has a theoretical net charge of zero when the ph is above a protein's pi it will carry a negative charge in case the ph is below a proteins pi it will be positively charged proteins differ in their pi and this have different charges in the same environmental ph placing them into an electric field leads the proteins to move in different directions positively charged proteins move towards the cathode negatively charged proteins towards the anode and if the pi equals the ph they did not move at all one example of this technique is native page in native page proteins are separated in a polyacrylamide gel the ph of the gel is set so that all proteins will carry a negative net charge therefore they will migrate to the positive electrode proteins are separated by the charge density which is the charge to mass ratio size which is their molecular weight and shape the greater the charge density of a protein the quicker it will migrate through the gel however larger proteins which have a higher molecular weight will find it harder to move through the gel and thus move slower furthermore if the protein has an elongated as opposed to a more global shape it will also migrate slower through the gel the target protein band can then be cut out of the gel the technique has a good separation effect but can only treat small samples it is therefore often used to validate other protein purification methods other methods that can be used to make sure that your protein purification approach was successful are scs page western blot and spectrophotometric essays we hope that this video was useful to you if you have any questions or feedback please leave it in the comments below and if you would like to see more videos like this one don't forget to subscribe to our channel