Unique among chromatographic techniques for protein purification, gel filtration separates proteins based on the relative size of their molecular masses. In contrast to conventional filtration, no protein remains after passing through the gel filtration column. The advantages and disadvantages of gel filtration are obvious; the advantage is that the binding of friable proteins to the chromatographic fixation does not disrupt its function; the disadvantage is that the lack of binding to the gel limits the resolution of the chromatography.
Authors: Burgess et al., Translated by Vivian Chen, this experiment is from "Protein Purification Guide".
Operation method
Gel filtration experiment Move manipulate The elution profile obtained using molecular sieve chromatography is shown in Figure 23.1 A. The zero elution volume is the volume of the sample that is sampled on the chromatographic stationary phase. The elution volume of molecules that cannot enter the medium is defined as the void volume V . which represents the volume outside the particle. The volume of molecules that can enter the medium is defined as the total volume %, which represents the sum of the internal and external volumes of the particle. The elution volume in between is V e. The partition coefficient is K av. Protein molecular mass determines the partition coefficient, and the semi-logarithmic curve between the two is shown in Fig. 23.1B. Separation of proteins based on the magnitude of the molecular mass is best achieved in the central linear region of the above S-shaped curve, which involves a K av value of 0.2 to 0.8. This range can be used as the separation range for the molecular exclusion mediator. In the separation range, the larger the slope of the S-shaped curve, the larger the resolution of the medium. Accordingly, when separating proteins with similar molecular weights, it is most suitable to use a medium with a narrower separation range. Less than 10 proteins can be separated from the effluent by any one sieve column. This low resolution occurs both because no protein is retained in the column during the analytical process and because of the undesirable flow around the particles. Therefore, if the target protein is of greater or lesser molecular weight than the majority of the protein in the mixture, the analytical purification (multiplicity) of the sieve may be significantly improved. Since this is not universally true, researchers can only expect a modest increase in purification (multiplicity). Therefore, it is sensible to place the sieve analysis as late in the purification program as possible, when the amount of other proteins is low and the previous steps have separated the protein mixture using methods of completely different characteristics. For example, the separated component that has been combined by ion exchange chromatography is probably still a mixture of proteins that have the same net charge but different molecular masses. The properties of some of the traditional high-efficiency sieve media are shown in Tables 23.1 through 23.4. It should be noted that suppliers use a variety of terms and abbreviations in their catalog references, including gel-filtration chromatography (GFO), gel-permeation chromatography (GPC), and size-exclusion chromatography (SEC). exclusion chromatography (S E C ). Traditional media are characterized by their economy and slow flow rates. These media are often sold in bulk form, allowing the researcher to fill any size column according to the volume of sample to be separated. The normal flow rates are shown in Table 23.2, with the bulk flow rate being the column cross-sectional area (cm2 ) multiplied by the linear flow rate (mIn/h). The flow rate of a packed column is approximately 5 times the flow rate during the chromatographic separation. High-performance media are characterized by ease of use, high flow rate, and high cost. These media are often sold as packed columns, and researchers can usually use existing high-efficiency chromatographic methods for purification. The smaller analytical columns are about 8 mm x 300 mm and normally have a protein load of a few milligrams and a flow rate of about ImL/million. Larger preparative columns are usually filled with particles of 30 um in diameter. 20 mm X 300 mm format columns have a protein load of 10 to 100 m g and an operating flow rate of about 5 mL/million. For very large columns, the protein load can be up to 2 g and the flow rate up to 30 mL/million. The concentration of protein in the sample should not exceed 50 m g/m L, and the clarified solution obtained after centrifugation should be used. If necessary, the sample should be taken at a low flow rate to prevent the formation of insoluble particles. Since the solvent is used after pretreatment of the proteins in the chromatographic separation process, the use of the solvent is irrelevant. There is a wide range of solvents that can be used in the columns, satisfying only the system-specified parameters, pH, temperature, etc., as shown in Table 23.1. However, proteins and mesophases can be combined by electrostatic or van der Waals action. In order to minimize the bonding, the ionic strength of the solvent used should not be less than 0.2 mol/L. The ionic strength of the solvent used should not be less than 0.2 mol/L. Since most proteins are inherently resistant to degradation at room temperature, the solvents used should have a solvent strength of no less than 0.2 mol/L. Chromatography columns can be filled in glass cylinders with simple solvents such as 0.1 mol/L NaCl equilibrated with 0.02% sodium azide to prevent microbial growth. The preferred storage solvent for filling columns in stainless steel cylinders is methanol to avoid accelerated corrosion of the container by the salt solution. In order to optimize the purification effect (multiplicity) of molecular sieve chromatography, it is necessary to use the medium that most easily separates the target protein. Similarly, primary sieving can be used to estimate the molecular mass of the target protein, as well as to differentiate the molecular masses of other proteins. In addition to protein samples, tools used to perform primary sieving include molecular sieve chromatography columns, distribution collectors, total protein content determination instruments, target protein content determination instruments, and protein molecular mass standard mixtures. Determination of total protein can be done by UV spectrophotometry or colorimetric methods (see Volume 182, Chapter 6 by Stoscheck). The concentration of the protein sample used for upsampling must be sufficiently large for the functional detection of the target protein in the eluate fraction to be credible. It can be assumed that the concentration of the target protein is diluted by at least an order of magnitude after chromatographic separation. Mixtures of protein molecular mass standards, commonly referred to as gel filtration standards, can be purchased from a number of vendors including Bio-Rad Laboratories (Richmond, CA), Pharmacia LKB Biotechnology (Piscataway, NJ), and Sigma Chemical (St. Louis, MO). Louis, MO). Such mixtures for calibration contain a number of identified proteins whose respective molecular masses and fractions are known and have defined V. and Vt. Researchers can also choose to If efficient molecular sieve analytical columns and chromatographic methods are readily available, primary sieving will be quick and easy. The chromatographic column used for primary sieving should have a wide separation range. A guard column should be placed in front of the analytical column to trap previously unnoticed particulate matter. Sampling can be done by injection, which ensures that the sample volume is minimized and is also suitable for the analysis of target proteins in the column effluent. If the absorbance of the solvent permits, an absorbance flow detector should be used and the detection wavelength should be set at the more sensitive 225 nm to monitor the protein concentration in the effluent, or alternatively at the relatively less sensitive 280 nm. If an absorbance flow detector is not available, the effluent fraction should be collected and analyzed for total and target protein. Finally, the gel-filtered standard mixture is injected and the effluent is monitored at the same wavelength. Comparison of the elution profiles of the gel filtration standard mixture, the total protein in the sample, and the target protein facilitates the selection of media with which to optimize the purification efficiency (multiplicity) of the molecular sieve chromatography. If high-efficiency analytical columns are not readily available, then conventional media with a wide separation range must be used for primary sieving. Generally, media that can be easily filled into columns should be selected. The filling of chromatographic columns with conventional media is described in more detail below. Similarly, primary sieving using gel-filtered standard mixtures, elution profiles of total sample proteins and target proteins are used to determine the media to be used for optimal molecular sieve chromatographic purification. The volume of conventional media used for protein purification should generally be 30 to 100 times the volume of the sample to be separated. Suspend the desired amount of media in the chromatography solvent and adjust the temperature to the conditions required for the separation operation. The volume of the suspension should not exceed twice the expected volume of the filled column. Gently shake the suspension to remove fine particles and aspirate the supernatant until approximately 90% of the particles have settled. Finally, place the resuspension under negative pressure to reduce the volume of dissolved air. Both filter flasks and laboratory aspirators can be used for this purpose. If the medium is in the form of a dry powder, it should be dissolved in the chromatographic solvent before removing fine particles. The medium can be swollen at room temperature or at 100° C, depending on the time available to the researcher. As shown in Table 23.2, it is faster to swell the media at 100° C without destroying the media. Chromatography column preparation should be carried out in a commercially designed or improvised laboratory cylinder of glass or clear plastic. The ratio of length to diameter of this cylinder is typically 20 to 100. The following procedure can be used for ad hoc fabrication. A thick rubber stopper with a narrow end and short length can be placed at the bottom of the column and surrounded by a capillary tube. Place the cylinder vertically and position it securely where the chromatographic separation will be performed. Insert the rubber stopper into the bottom of the end of the cylinder. A short, bendable tube is connected to the protruding glass tube and a clamping device is attached to the tube to control the flow rate of liquid through the cylinder. A mesh of amide fiber or PTFE resin is placed inside the cylinder and pushed to the bottom so that it is in contact with the rubber stopper. The clamping device is closed and the medium suspension is poured into the cylindrical column. The excess suspension is located in a container with an outlet and switching device at the bottom, e.g. a dispensing funnel, and the outlet is connected to the top of the cylinder with a bendable pipe and a rubber plug with a short glass tube hole. In this type of assembly, the surface of the suspension in the dispensing funnel and the bendable pipe connected to the bottom of the cylinder are closed. The flow rate of the liquid is controlled by the relative height to the dispensing funnel of the cylindrical column. The flow rate during column filling can be 5 times the flow rate shown in Table 23.2. Once the column has been filled to the required height, the clamps and switches can be closed, the excess media suspension removed, and the dispensing funnel used as a vessel for the flow of chromatography solvent through the column. The top of the column should be maintained at a height of a few centimeters to cushion the impact of the addition of the solvent and to keep the top of the filled column intact. It is important not to allow the filled column to dry out, as this can cause channels to form in the column, which can severely affect the separation of proteins. To load the sample, turn off the switch, remove the rubber stopper from the tip of the cylinder, and drain the solvent collected at the tip of the cylinder through the column until the solvent soaks under the tip of the loaded column. Afterwards, close the clamp and carefully add the sample or standard solution to minimize impact on the top of the installed column. Next, open the clamp and allow the sample solution to enter the column until it is just below the top of the column. Close the clamp again and add a small amount of solvent for dialysis, taking care to minimize the impact on the top of the loaded column. Once the solvent is in the column, close the clamp again and add more chromatography solvent to create the desired solvent height. A dispensing funnel is used to supply the chromatography solvent and is connected to the top of the column by a bendable tubing seal. Finally, the height of the dispensing funnel is adjusted and the desired flow rate is maintained. The absorbance of the post-column effluent can be continuously monitored at the desired wavelength using a flow controller. It is important that the tubing material at the bottom of the column and the flow optics in the monitor have small diameters so that convective mixing of the liquid in the column can be prevented. It is also important that the bendable tubing used should be of a material that has no effect on the UV absorbance value of the chromatography solvent. Alternatively, the post-column effluent can be collected directly in a graded collector, after which the separated fractions can be analyzed for total and target proteins. A drop counter is the ideal tool for this purpose. Sieve analysis with traditional media can easily be enlarged, and the column size can be increased to accommodate the sample size to be separated and purified. Where the sample size is very large, repeated analysis is the preferred method of dealing with very large column sizes. Table 23.4 shows the high performance columns that are available in semi-preparative or preparative sizes, and some suppliers also provide bulk media for researchers to fill columns. Although these larger high-performance columns are very expensive, they can save the researcher a lot of time and have many different uses, so this investment is worthwhile in the long run. Low resolution Low resolution is a common perception amongst users, as sieve analysis inherently suffers from poor resolution. Nonetheless, it is possible to improve resolution by changing some of the operating parameters. First, since flow rate is inversely correlated with resolution, lowering the flow rate will increase the resolution. Secondly, the use of smaller diameter particles can increase the resolution. Finally, the use of a medium with a narrower separation range may help. Slow flow rates Chromatographic Peak Dump Chromatographic peak tilt is mainly caused by incorrect sampling. For traditional columns, inert colored components such as glucose blue or potassium dichromate may be added to the sample to monitor the quality of the sample. If the sample appears irregularly on the column, asymmetrical peaks will appear in the elution profile. The sample feeder of a high-efficiency column can be removed and cleaned. Trailing peaks are generally caused by the adsorption of protein to the mediator. This can be improved by using more soluble salts as the main ionic component of the analyte, e.g. sodium perchlorate instead of sodium chloride. If tailing occurs when using a high-efficiency column, it may indicate that the encapsulating material for the silicon dioxide particles is too depleted and the column needs to be replaced. Skewed peaks can also be caused by a reversible equilibrium between different states of protein aggregates. For example, hemoglobin may show a dynamic equilibrium between dimers and tetramers. Since polymerization revolves around the molecular mass of protein, the mesomers must contribute to dissociation and the chemical equilibrium to polymerization. For this kind of state-of-motion exchange, these opposing forces lead to the appearance of oblique peaks. Changes in the pH, temperature, or chemical composition of the analyte solvent may also change the chemical equilibrium, leading to a significant increase in only one polymer. Loss of target protein There are at least two reasons for this phenomenon. The first reason is that the target protein is moderately bound to the column media and the elution peaks are broad, resulting in the eluted target protein appearing after V t , making it difficult to distinguish it from the baseline noise. If this occurs, a protein solubilizer, such as a nonionic detergent or an appropriate concentration of protein denaturant, should be added to the chromatography solvent. The second reason is that the functional protein complex breaks down into discrete proteins of different molecular masses, which themselves cannot maintain their original function. Mixing the different components may help the complexation of the component proteins and thus restore their function. For more product details, please visit Aladdin Scientific website.
The partition coefficient is K av, and the relationship is shown in equation (23.1).
Since most proteins are relatively stable at room temperature, and any proteolytic enzymes present in the protein sample will catalyze peptide hydrolysis, the rate of hydrolysis can be slowed down by lowering the temperature. However, protein hydrolysis at the purification stage has become an increasingly important issue, as the target protein is enriched and the substrate for protease action becomes richer for the protease. In some cases, we also need to add more expensive protease inhibitors or effectors to the chromatography solvent in order to maintain the function of the target protein . Alternatively, it may be economically feasible to equilibrate a column volume with a solvent containing the expensive inhibitor component and then sample the protein. After sampling, there is no need to add the expensive inhibitor component to the solvent.
already purified components as calibration mixtures. Dextran blue (blue dextran) and DNA restriction fragments are commonly used to determine V. It is important not to utilize small molecules for this purpose. It is important not to utilize aromatic or heterocyclic compounds with small molecular masses for the determination of Vt, as these molecules are particularly susceptible to reversible adsorption with molecular sieve chromatography media.
Slow flow rate is usually caused by particulate matter in the sample clogging the filtration device or analyzing medium. Treatment: Reverse flow cleaning of the column with solubilizing reagents such as ionic or non-ionic detergents, protein denaturants such as urea or guanidine hydrochloride, organic solvents such as methanol, or short-term exposure to strong acids or bases to ensure stability of the media. If the above procedures are not effective, it is recommended that the columns be disassembled, cleaned separately, and reassembled for traditional media. For high-efficiency columns, it is recommended that the columns be sent to Phenomena or other suppliers for reimbursable cleaning and reassembly, or replaced with new columns in a simple manner. Laboratories with column reassembly facilities can also clean and reassemble high performance columns themselves.