The experimental step of measuring protein molecular weight by gel layering method.

related topics . The experimental method of molecular weight determination and the strong weapon I, the experimental purpose
1. To understand the principle of gel analysis and its application.
2. Through the training
measuring
molecular weight of proteins, the initial knowledge of gel
and layering technology
.
II, experimental principle
gel layering is also known as anti-layering, gel
filtration
, osmosis or molecular sieve layering. It is widely used in separation, purification, concentration of biological molecules and desalination, deheated sources and so on, and the determination of the molecular weight of proteins is also one of its important applications. Gel is a kind of insoluble beaded particle material with three-dimensional mesh structure and porous. It is used to separate substances, mainly according to the porous gel on different radius of protein molecules (near the spherical) have different emission effects. That is, it is separated and purified according to the physical properties of molecular size. For a certain type of gel, some large molecules can not enter the inside of the gel particles and are completely blocked outside, can only flow out of the column along the gap between the particles, while some
small molecules
are not blocked, can spread freely, penetrate into the inside of the gel sieve holes, and then be taken away by the outflow of semen. The smaller the molecule, the deeper it gets into the inside of the gel, and the more distance it travels, the smaller the molecule finally flows out of the column, and the longer the large molecule flows out of the column first. Some medium-sized molecules are between large and small molecules and can only enter a portion of the gel's larger pores, i.e. partially blocked, so the order in which these molecules flow out of the column is also between large and small molecules. So the sample after gel analysis, the molecules will flow out in order from large to small, to achieve the purpose of separation.
gel
separation
the principle schematic see "Figure 3-5" on page 64.
For any isolated compound in the gel
clysis column
in the range of 0 to 100%, the degree of displacement can be expressed by the effective distribution coefficient Kav (separation compound in the volume of inner and outer water), Kav value size and the total accumulation of the gel column bed (Vt), outer water volume (V0) and the escape volume (Ve) of the separator itself:
Kav s (Ve-V0)/(Vt-V0) ----------- (1)
Vt and V0 are constant values under limited layering conditions, while Ve changes with the amount of separator
molecule
. The molecular weight is large, the Ve value is small, and the Kav value is small. Conversely, the molecular weight is small Ve value is large, Kav value is large.
Figure 3-6 on page 65 shows a diagram of the gel itself (base) volume (Vg), the outer water volume (V0), the inner water volume (Vi), and the total column bed volume (Vt) in the gel column.
3-7 on page 65 shows several of the tops in the gel analysis column.
effective distribution coefficient Kav is an important parameter to judge the separation effect, and also a basis for determining the molecular weight of proteins. Under the same layering conditions, the greater the difference in the kav value of the separated substance, the better the separation effect. Conversely, the separation effect is poor or cannot be separated at all. In the actual experiment, we can measure the values of Vt, V0 and Ve to calculate the size of Kav. For a particular type of gel, Kav is linearly related to logMw (Mw represents the molecular weight of the substance) within a certain molecular weight range:
Kav s-b logMw s C --------- (2)
where b, C is constant.can also be obtained:
Ve s-b'logMw s C' --------- (3)
where b', C' is constant. That is, Ve and logMw are also linear. We can measure the molecular weight of other unknown proteins by separating a variety of proteins with a variety of known molecular weights on a gel column and drawing a standard curve based on the linear relationships mentioned above.
III, equipment and
reagents
(i) equipment
1. Glass laminate column ((20mm×60cm)
2. Constant current pump (or lower constant pressure reservoir bottle)
3. Automatic partial collector
4. UV ionography photomedia
5.100ml reagent bottle
6. 1000ml tube
7. 250ml beoth
8. 50ml, 100ml beabus
9. 10ml (or 5ml) scale test tube
(ii) reagent
1. Standard protein
(1) cow
serum
albumin: Mw s 67,000 (Shanghai
bio-chemical
institute)
(2) chicken egg serum protein: Mw s45,000 (SIGMA Corporation of the United
(3) Pancreatic Lactase Original A:Mw-24,000 (SIGMA Corporation of the United States)
.(4) Lysolysase: Mw s 14,300
2. Unknown protein samples: prepared by the laboratory
3. 0.025M KCl-0.1M HAC (acetic acid) (desalination 1000ml)instruments and reagentsinstruments: stratospheric columns, constant current pumps, ultraviolet detectors, automatic partial collectors reagements samples to be tested: insulin, bovine serotoninblue glucosin 2000; Sephardex G-75; wasting agentexperimental step 1. gel swelling gel particles require a relatively uniform size, stable flow rate, and better results.Take glucosaccharide Sephardex G-75 dry powder, add excessive distilled water room temperature fully soluble for one day (the swelling time varies depending on the intersection of the gel), or boiling water bath swelling for 3 hours, which can greatly shorten the swelling time, and can kill bacteria and mold, and can drain the bubbles inside the gel. Take care not to stir too much during the swelling process to prevent particles from breaking. To dissolve the balance after the use of pouring method to remove the small particles that are not easy to sink, and finally the gel after decompression pumping to remove the bubbles, you can prepare to load the column.. 2. Before loading the column the column, pour out too much of the solution on top of the gel and install the column vertically. Close the outlet, add about 1/3 column volume of the desalination to the column tube, and then stir, the thick slurry-like gel continuously poured into the column, so that it naturally subsides, until the gel sinks about 2-3cm, open the column exit, adjust the appropriate flow rate, so that the gel continues to sink, the glue surface to be collected rises to about 5 cm away from the top of the column when the stop loading column, close the outlet. The mounting column requires continuous, uniform, bubble-free, and no "pattern". is connected to the constant current pump, and the outlet of the constant current pump is connected to the analysis column. Balance the column bed with an escape solution of 2-3 times the volume of the column bed, and then place a filter paper or nylon filter cloth on the gel surface to prevent the gel from being flushed up at the time of the sample addition in the future, and always maintain a liquid on the upper end of the gel. . 3. The upper column of the sample is one of the keys to the success or failure of the experiment, if the sample is diluted or the upper column is not equal, it will spread the zone belt and affect the effect of layering. Try to keep the bed as stable as possible. First open the exit of the column, wait for the column to drain the liquid flow from the surface of the bed 1 to 2mm, close the exit, with a dropper to slowly add 1 ml sample to the surface of the column bed, should avoid the bed surface gel flushed up, open the outlet and start to calculate the outflow volume, when the sample into the bed close to the surface of the bed 1mm closed the exit. According to the sample operation, rinse the pipe wall 2 times with a small amount (about 1 ml) of semen. Finally add a small amount of semen on the gel, so that the surface of the bed 3 to 5 cm, tighten the upper mouth screw cap, column inlet connected to the constant flow pump, adjust the flow rate, to each tube 3 ml/10min flow rate to start washing. The volume of the upper size, the analysis amount is 1-2% of the volume of the column bed, and the spare amount is 20% to 30% of the volume of the column bed. . 4. Collection and identification with an automatic partial collector to collect the outflow, 4 ml per tube, UV detector 280nm wavelength detection, the highest OD value when the volume is the absorption peak of the excrucation volume Ve. . 5. Treatment of gel column gel after use, repeatedly washed with distilled water after preservation, if there is a color or relatively dirty, can be washed with 0.5mol /LNaCl. Short-term can be stored in the water phase, add
preservatives
or
heating
sterilization and preservation at low temperatures. It is recommended to
in
dry state for a long time. the of molecular weight are calculated, the standard curve method is generally used. As long as several standard proteins are measured relative to the molecular mass of Ve, and their relative molecular mass logMr is used to map Ve in a straight line, and then the Ve of the sample to be tested can be measured, its relative molecular mass can be determined from the graph. note the connectors can not leak gas, continuous use of small latex tube should not be broken, otherwise cause air leakage, leakage. Note that the exhaust pipe in the constant pressure bottle should be liquid-free, and with the flow of solution in the lower mouth of the column constantly bubble production, indicating that the constant pressure bottle does not leak. During operation, the fluid surface in the stratum column is constantly decreasing, indicating that there is a leak in the entire system, which should be carefully examined and corrected. always keep the liquid surface in the column higher than the gel surface, otherwise the moisture evaporates and the gel drys out. It is also necessary to prevent the liquid from running dry, so that the gel mixed into a large number of bubbles, affecting the flow of the liquid in the column, resulting in a bad separation effect, had to reload the column.
.