Gel filtration chromatography is an important protein purification technique, also known as size exclusion, gel exclusion, molecular sieve, or gel filtration chromatography This method utilizes hierarchical separation without the need for chemical binding of proteins, which significantly reduces protein loss and inactivation due to irreversible binding. In addition, this method can be utilized to change the buffer for proteins or to reduce the ionic strength of the buffer. It is not possible to generalize when to use gel filtration in a protein purification operation; sometimes gel filtration is applicable when purification reaches a certain step. For a protein with a large molecular weight (e.g., more than 100 KD), the first step in the purification process may be gel filtration chromatography. By using the appropriate column packing, the target protein will be present in the front portion of the collection, while all of the small molecular weight proteins remain on the column, thus completing the first step of the purification process. If ion exchange chromatography has been used to partially purify the proteins, then gel filtration is the ideal next step in the purification process, as the two separation methods are complementary in principle. Selecting a narrow separation range packing material for a protein of similar molecular weight gives a better separation, whereas separating a protein of unknown molecular weight requires a wide separation range packing material. For a protein of high purity, gel filtration chromatography can be used to detect whether the active form is a monomer or an oligomer. In summary gel filtration is a separation method that can be used at any stage of protein purification. Source: Handbook of Protein Technology
Operation method
Gel Filtration Chromatography
Principle
Gel filtration chromatography achieves the separation effect according to the different sizes of protein molecules. The gel filtration packing contains a large number of micropores, which only allow the buffer and small molecular weight proteins to pass through, while the large molecular weight proteins and some protein complexes are blocked out. As a result, high molecular weight proteins flow through the interstices of the packing particles and are eluted earlier than low molecular weight proteins. The largest protein molecules flow out of the column first because they pass through the smallest volume before reaching the bottom of the column. Medium-sized proteins can enter the larger pores of the packing molecules, so they reach the bottom of the column later, while small proteins, which can enter all the pores of the packing, have the largest passing volume and therefore reach the bottom of the column last.
Materials and Instruments
Protein solutions Move 1. Filling of gels 1.1 Dissolving the gel If the gel is a dehydrated dry powder (e.g. Sephadex) it needs to be solubilized before use. 1.1.1 Add 10 parts of buffer to one part of gel. In principle, the gel particles should be placed in the elution buffer. 1.1.2 Mix on a shaker or by hand stirring, avoiding the use of electric stirrers. This is because mechanical stirring forces can cause the gel particles to break into fragments and these fine fragments will interfere with the chromatography. If fine gel fragments appear the gel slurry can be suspended, and after the gel particles settle, remove the supernatant and repeat several times until the liquid phase is no longer turbid. 1.1.3 Natural swelling requires 24 h to several days, in order to accelerate the swelling, can be used to heat the swelling. That is, in the boiling water bath will gradually warm the gel paste to nearly boiling, which can accelerate the swelling equilibrium, usually 1 ~ 2 h can be completed. This method not only saves time, but also eliminates air bubbles and sterilization. 1.1.4 Whether the gel needs to be swollen or not, in order to prevent air bubbles affecting the chromatography, the gel slurry needs to be pumped with a water pump or a vacuum pump for 1 hour. 1.2 Loading column 1.2.1 Install the column vertically in a place without direct light and air convection to prevent air bubbles in the column due to temperature change. It is usually placed in a special chromatography cooler; 1.2.2 Make a gel slurry of 3/4 volume of gel and 1/4 volume of buffer before loading the column; 1.2.3 For frequently used columns, it is recommended that a commercial mobile phase adaptor (flow adaptors) be used. It protects the surface of the column bed and also prevents large particles in the sample from mixing into the gel, thus prolonging the life of the chromatography column; 1.2.4 Add buffer to the chromatography column and close the outlet of the column when buffer flows out. This removes air bubbles from the voids below the support filters in the chromatography column; 1.2.5 Pour the gel slurry along one side of the column or along one glass rod; this must be done at one time or the column bed will not be homogeneous; particular care must be taken not to create air bubbles during the column loading process; if bubbles appear early in the column loading process, a glass moisture rod can be used to agitate the column so as to dislodge the bubbles; 1.2.6 After the gel has been loaded to the bottom of the column, open the bottom outlet to speed up the process of loading the column and subsequently add more buffer; 1.2.7 connecting the chromatography column to the peristaltic pump and reservoir and washing the column with several times the volume of buffer in the column bed to stabilize and equilibrate it; 1.2.8 SephacrylHR and Superose gels are loaded onto the column in two steps at different flow rates (see handy instructions for details); 1.2.9 Care must be taken that the column buffer is not allowed to run dry at any time, as this will prevent normal chromatographic separation. 1.3 Inspection of the chromatographic bed Because air bubbles and column inhomogeneities significantly reduce chromatographic discrimination, the homogeneity of the chromatographic bed must be carefully checked prior to chromatography. 1.3.1 Observe the chromatography column for air bubbles and cracks by eye to light. 1.3.2 The homogeneity of the chromatographic bed is further checked accurately with 2 mg/ml blue dextran solution. The blue color is removed by using 1% of the bed volume and twice the amount of eluent. A common eluent is 0.02 mol/L sodium chloride. If it is necessary to avoid the presence of salt in the connection experiments, the blue color can be eluted with additional water. If trace amounts of blue dextran are adsorbed on the gel surface, serum albumin solution is often used to elute. The blue color of the blue dextran is evident and if the column is well packed, the blue zone can be seen to pass evenly and smoothly through the gel without leaving any streaks. In the case of Bio-Gel P columns, hemoglobin and ferritin can also be used instead of blue dextran as a standard reference for examination. This is because they are also colored proteins. Blue dextran can also be used to measure the volume of water outside the chromatography column. The external water volume is the volume of eluent required to elute large particulate matter from the column that cannot enter any of the micropores in the gel. The external water volume is the smallest necessary volume in which proteins may be eluted from the column, and it can be used as a relative standard for predicting the amount of eluent required for other proteins. 2. Sample Loading and Elution 2.1 Sample Loading 2.1.1 Protein samples should be highly concentrated (10-20 mg/ml) and as small as possible (1% to 5% of the column bed volume is appropriate). Sample volumes higher than 5% of the column bed volume will reduce the effectiveness of the separation. The low volume of the column bed does not improve the separation effect, in addition, the viscosity of the sample should not be too high, otherwise it will make the chromatographic zone unstable and the flow rate is irregular, resulting in zone broadening or distortion. 2.1.2 Before sampling, the sample should be filtered through a membrane with a pore size of 0.2 um or centrifuged at 10 000×g for 5 min, in order to remove the residue that interferes with chromatography. 2.1.3 The buffer chosen should facilitate the maintenance of protein activity and prevent non-specific protein-protein or protein-gel interactions. Generally, low ionic strength salt solutions (20-100 mmol/L) are sufficient to block non-specific ionic interactions. In some cases, hydrophobic interactions between proteins and gels may occur and high ionic strength salt solutions should not be used. After the column has been equilibrated, close the outlet when the equilibrium solution has flowed 1 to 2 mm below the bed surface. Do not allow the column to run dry; it must be reloaded when dry; When the sample is added to 2-3 cm above the surface of the column bed with the sampler, connect the constant pressure sample bottle and open the outlet to let the sample seep into the gel. After adding the sample, wash the surface with a small volume of eluent 1 to 2 times to dilute the sample as little as possible. When the sample is nearly dry, add the eluent as carefully as the sample, and when it reaches 2-5 cm above the surface of the bed, connect the constant-pressure eluent bottle and start chromatography. All of the above steps must be performed with constant attention to the homogeneity of the chromatographic surface. 2.2 Sample Elution 2.2.1 The buffer is flowed through the column and eluted until the target protein is detected; 2.2.2 The flow rate of the column is usually controlled by a peristaltic pump. The pressure of the pump used should not exceed the tolerance of the gel; 2.2.3 Since gel filtration chromatography is often performed for more than a few hours, in order to prevent the column from running dry, when a peristaltic pump is not in use, a hose can be added between the reservoir bottle and the upper inlet of the column at a length equal to the outlet of the column, so that the buffer solution flows through the hose and then reaches the column. 2.2.4 Ideal sample recovery can be over 85%; 2.2.5 Low flow rates improve peak resolution; 2.2.6 With regard to protein molecular weight determination (mainly globular proteins under non-denaturing conditions) this can be solved by gel filtration column chromatography of protein standards. However, this method cannot be used for molecular weight determination under denaturing conditions. A standard curve is obtained by plotting the logarithm of the molecular weight of a standard protein against its elution volume. The molecular weight of the unknown protein is then plotted against the eluent of the standard protein to determine the corresponding molecular weight. 3. Regeneration and preservation of chromatography columns Since the gel is unlikely to bind protein, it is not difficult to remove protein from the gel. Cleaning with dilute sodium hydroxide or a non-ionic detergent usually removes most of the bound material. However, the most effective way to avoid contamination of the column is to filter the sample prior to injection and to run the column through a freshly filtered buffer. If some of the contaminants cannot be removed by the standard procedure above, the resin can be poured from the column and reloaded after special treatment as described below. Hydrophobic proteins can be removed by overnight elution of the column with 24% ethanol or 30% acetonitrile; hydrophilic proteins can be removed by elution with 30%-50% acetic acid or by incubating the gel in the column overnight with 1 mg/ml pepsin (dissolved in 0.5 mol/L NaCl and 0.1 mol/L acetic acid). Treatment with protein hydrolase breaks down the remaining traces of pepsin in the gel. Nucleic acids are removed by washing with TE buffer, (10 mmol/L Tris-HCl, 1 mmol/L EDTA pH 8.2) or treatment with nuclease. Lipids can be removed by eluting the column overnight with a nonionic detergent such as 0.2% to 1% NP-40 or Lubrol. Chromatography columns should be stored in a buffer containing a preservative and kept at a low temperature to help prevent microbial growth. The addition of 0.02% sodium azide to the buffer usually prevents microbial growth, and Sephacryl HR or Sepharose CL can be stored in 20% ethanol. Some gels can be autoclaved to prevent microbial growth. Caveat 1. When adding samples, do not use ordinary droppers, but use droppers with larger lower mouths, so as not to stir up the surface of the column bed by the pressure generated by the dropper head. 2. Sample filling should be done skillfully and carefully to avoid mixing the surface of the bed. In the equilibrium of the column bed surface will often appear concave phenomenon, so we must check whether the surface of the column bed is uniform, if it does not meet the requirements, can be used to fine glass rod to gently stir the surface layer, so that the gel naturally settled, the surface is uniform. 3. The method of adding samples is to utilize the principle of stratification of two liquids with different specific gravity, add the high specific gravity samples into the low specific gravity eluent on the surface of the bed, and the samples will sink slowly and evenly on the surface of the column bed. The sample then sinks slowly and uniformly onto the surface of the column bed. The outlet is then opened to allow the sample to penetrate into the chromatography bed. If the specific gravity of the sample is not high enough, glucose or eluent can be added to the sample to achieve a final concentration of 1%. For more product details, please visit Aladdin Scientific website.
Buffers Blue dextran solution Ethanol Acetonitrile Acetic acid Pepsin
Low Pressure Chromatography System