Molecular weight determination of proteins by gel filtration chromatography experiment
Molecular weight determination of proteins by gel filtration chromatography experiment
Gel filtration is also known as exclusion chromatography, molecular sieve chromatography and gel chromatography. The purpose of this experiment is to understand the basic principles of gel filtration chromatography to determine the molecular weight of proteins and consolidate the operation of column chromatography.
Operation method
Gel Filtration Chromatography
Principle
Gel Filtration is also known as Exclusion Chromatography, Molecular Sieve Chromatography and Gel Chromatography. Gels are generally solid substances formed by the condensation of a colloidal solution of dextran, which has a very fine porous mesh structure. The mechanism of gel chromatography is the molecular sieve effect, when the gel is loaded into the column, the volume of the column bed is called the "total volume" ( Vt ), which consists of Vo, Vi, Vg three parts, Vt = Vo + Vg, where Vo is the "volume of the outer water," or Vo is the "external water volume", or "pore volume", i.e., the volume of the aqueous phase that exists between the pores outside the gel particles in the column bed; Vi is the "internal water volume", i.e., the volume of the aqueous phase inside the gel particles, which is equivalent to the volume of the solid phase in general chromatography, and can be obtained from the weight of dry gel particles and the weight of absorbed water. It can be obtained from the weight of dry gel particles and the weight of absorbed water; Vg is the volume of the gel itself, Vt - Vo = Vi - Vg. The "elution volume" (Ve) is the volume that flows out from the beginning of sample addition to the maximum concentration of the component (peak), which is related to Vo and Vi as follows: Ve = Vo - Kd Vi , where Kd is the partition coefficient of the sample in two phases, i.e., the partition coefficient of different molecular weights, which is the distribution coefficient of the sample in two phases. Kd is the partition coefficient of the sample in the two phases, i.e., the partition coefficient of solutes with different molecular weights inside and outside the gel, which is only related to the size of the molecules of the separated substances and the distribution of the size of the pores of the gel particles, and has nothing to do with the shape of the column, and it is a constant for a specific substance. For a chromatographic column gel bed, as long as the experimental knowledge of a substance elution volume Vo, you can find its Kd value, the above formula can be rewritten as Kd = ( Ve - Vo ) / Vi, in which Vo can be used to find the large molecules that are not retained by the gel solution, such as blue dextran-2000 ( MW = 2000000 U ), hemoglobin, India black ink, etc.; Vi can be used to find the g-Wr ( g is the dry gel ), and the gel particles can be used to obtain the distribution coefficient ( g is the dry gel size distribution ). Vi can be obtained from g-Wr (g is the weight of the dry gel, Wr is the amount of water absorbed by the gel, expressed in ml/g). The gels currently used are dextran gels, polyacrylamide gels, agarose gels and composite gels composed of agarose and dextran. These gels have the following characteristics: they are chemically stable, do not react with the substances to be separated, have no or very few ion exchanger groups, and have sufficient mechanical strength. The porous granular gel made by cross-linking dextran and chloropropane oxide was used in this experiment. The larger the degree of cross-linking, the smaller the pore size, it is insoluble in organic solvents, can be quickly dissolved in water and electrolyte solution. Different types of dextran gels are indicated by the English G, such as G-25, G-50, G-75, G-100, etc., the latter number is the gel water absorption multiplied by 10. In practice, the ultrafine particles are mainly used in column chromatography with very high resolution, the fine particles are used in preparation, and the medium and coarse particles are used in the preparation of the column chromatography with a high flow rate at a low operating pressure. According to the principle of gel chromatography, the elution characteristics of the same type of compounds are related to the molecular weight of the components, and when flowing through the gel column, the molecules flow out in the order of molecular size, and the one with the larger molecular weight goes to the front. Usually Ka v ( Kd ) to the molecular weight of the logarithm of the graph can be a curve, called "selection curve", the slope of the curve is an important feature of the gel properties, within a certain range, the steeper the curve the better the grading. In the determination of molecular weight, it is appropriate to use the straight line part of the curve.
Materials and Instruments
Protein solutions Move 1. Slowly stir the Sephadex suspension, then slowly pour it into the pre-set column. The gel bed was made up to 2.5 cm×30 cm, and the top outlet tube was controlled by a screw clamp. The gel bed was made up to 2.5 cm×30 cm, and the top outlet tube was controlled by a screw clamp. The top outlet tube is controlled by a screw clamp. No air bubbles should be generated. When the gel has been completely settled, control the liquid level on the gel bed to about 5 cm. When the gel has completely settled, control the liquid level on the gel bed at about 5 cm. For more product details, please visit Aladdin Scientific website.
SephadexG-75 Tris-HCl dextran myoglobin mass DNP-aspartic acid glycerol
Chromatographic Columns
2. Next, add the substance to be separated to the upper end of the column (the liquid level at the top of the column bed is lowered until the gel is just exposed). Then, add the substance to be separated to the upper end of the column (the liquid level on the column bed is lowered until the gel is just exposed), and suck up the sample to be separated with a pipette. Pipette the sample to be separated and position the tip of the pipette approximately 1 cm above the gel bed, hold the pipette in place without changing its position, and then slowly and continuously remove the sample from the column. Hold the pipette in place without changing its position and release the sample slowly and continuously onto the gel bed. Remove the pipette. Remove the pipette and place a 10 ml tube in a portion collector to collect the solution from the lower end of the column. The flow rate is controlled to be 1 drop every 4 seconds (equivalent to 60 ml/h). 3.
3. When the sample solution has just completely flowed into the gel bed, add buffer very carefully to the top of the column to a suitable level. When the sample solution has just flowed completely into the gel bed, add buffer to the top of the column very carefully to a suitable level to ensure a steady flow rate. Be careful not to agitate the gel at the upper end of the column. The top of the column should not be stirred, nor should the top of the gel bed be left without sufficient buffer or even dried out. 4.
4. Collect 28 ml of effluent first, then start collecting at 4 ml per tube, successively collecting and numbering each tube. Collect and number each tube, and when all the colored material has eluted from the column, close the screw clamp to stop the elution. When all the colored substances were eluted from the column, the elution was stopped by closing the screw clamp. The absorbance values of each tube were then read at the corresponding wavelength. Dextran (650 nm blue); myoglobin (500 nm amber); DNP-aspartic acid (440 nm); and myoglobin (500 nm amber). Aspartic acid ( 440 nm yellow).
5. After elution of the last band (yellow), 1 ml of unknown sample solution was added to the column. After elution of the last band (yellow), 1 ml of unknown sample solution was added to the column, 28 ml was collected first, and then every 4 ml was collected continuously as described above. Start with 28 ml, and then collect continuously every 4 ml as described above. If the protein is colorless, read the absorbance at 280 nm. If the protein is colorless, read the light absorption value at 280 nm. Calculation of results
Calculation of Kd value: Use the light absorption value of each tube to graph the number of tubes (note that the first 7 tubes are numbered with 28 ml). The light absorption value of each tube is plotted against the number of tubes (note that if the first 7 tubes were collected in 28 ml at one time, the number of the first 4 ml tube should be 8), and the measured peak position should be labeled. The measured wavelength should be marked at the position of each peak, and a coordinate graph should be drawn to determine the exact position of each peak by observation or arithmetic averaging. The exact position of each peak should be determined by observation or arithmetic averaging. The formula for arithmetic averaging is: Peak midpoint=Peak number multiplied by the number of tubes in the peak. The formula for arithmetic averaging: peak midpoint = the number of each tube in the peak multiplied by the sum of the light absorption values of the consecutive tubes in the peak, divided by the light absorption value of the consecutive tubes in the peak. The midpoint of the peak is calculated as
This was also done for 1 ml of unknown sample. The Kd of myoglobin and the Kd of the unknown sample were calculated. Kd values for myoglobin and the unknown sample were calculated.
Estimation of molecular weights of myoglobin and unknown samples: Using the data in the table below, the Kd values of the known proteins (Kd) were calculated. Using the data in the table below, graph the Kd values (vertical coordinate) of the known proteins against lgMW, and then calculate the molecular weights of myoglobin and the unknown proteins. The molecular weights of myoglobin and unknown proteins were calculated.
Label the Kd and MW of the myoglobin and unknown samples at the appropriate places in the graph.
