Separation of proteins of different molecular weights - gel column layering method.

objectives require(1) to understand the principle and application of gel column analysis.(2) master the basic operating techniques of gel column analysis.principlegel layering, also known as gel
filtration
, is a method of layering materials separated by molecular weight. The method is to add the sample to a column filled with gel particles and then wash it off with a buffer. Large molecules can not enter the static phase in the gel particles, only remain in the flow phase between the gel particles, so at a faster rate first flow out of the column, and small molecules can freely enter and leave the gel particles, and quickly form a dynamic balance between the flow phase and the stationary phase, so it will take a long time to flow through the column bed, so that molecules of different sizes can be separated.used for gel filtration column analysis is a substance with a three-dimensional mesh structure, a consistent diameter of the sieve holes, and beaded particles. This substance can completely or partially block certain large molecules
compounds
outside the perforation, while for some small molecular compounds can not be blocked, but can allow it to spread freely in the perforation, penetration.
the degree to which any isolated compound is blocked by the gel sieve hole can be expressed by the distribution coefficient Kav (the ratio of the separated compound in the volume of inner and outer water). The size of the Kav value is related to the total product (Vt) of the gel bed, the volume of external water (Vo), and the exculation volume (Ve) of the separator itself, i.e.: Kav= (Ve-Vo)/(Vt-Vo)under limited layering conditions, Vt and Vo are constant values, while ve values vary with the molecular weight of the separator. The molecular weight of the separator is large and the Kav value is small;usually choose blue glucosaccharies 2000 as a substance to determine the volume of external water. The substance has a large molecular weight (2 million) and is blue, it is completely blocked in various models of glucosaccharol gels, and can be detected by the naked eye or spectrograph (210nm or 260nm or 620nm) with the help of its own color (i.e. Vo).
However, when measuring the molecular weight of
proteins such as
kinase , it is advisable to use blue glucosaccharin 2000 to determine the volume of external water, because it has adsorption effect on kinase, so sometimes with giant globulin instead. To determine the volume of internal water (Vi), it is available with ammonium sulfate, N-acetyl tyrosine ethyl, or other small molecular substances that are not adsorbed with the gel.this experiment used a mixture of hemoglobin (molecular weight of about 64,500) and nitrofluorobenzene (DNP-fish protein molecular weight of about 12,000) to achieve separation after analysis by Sephadex G-25.reagents
equipment 1, reagents 0.9% NaCl; 10% NaHCO3); 95% ethanol.pH 7.0 coagulation buffer (20mM sodium phosphate: 20mM hydrophosphate ddi sodium: 31mL: 69mL) 2, material hemoglobin solution (Hb): take anticoagulant (heparin) 2mL, centrifugal abandonment of the upper plasma. Use 0.9% NaCl to wash blood cells several times (reverse mixing, centrifugation, discarding the upper liquid), so that the centrifugal liquid after almost no pale yellow. Add 5 times the volume of distilled water to the washed red blood cells to shake well, centrifugal precipitation (broken cell membranes, etc.) is the Hb dilution backup. . DNP-fish sperm protein solution: take fish sperm protein 0.15g dissolved in 10% NaHCO3 solution 1.5mL (at this time the protein solution pH should be about 8.5 to 9.0). Another 0.15g of nitrofluorobenzene was taken and dissolved in 95% ethanol 3mL of microthermal, and dumped into the above protein solution immediately after its full dissolution. Bring the tube to a boiling bath for 5 minutes, paying attention to prevent ethanol from boiling and spilling. After cooling, 95% ethanol is doubled in volume, and yellow DNP-fish protein precipitation is visible. Centrifugation (300r/m) 5 minutes, discard the liquid, precipitation with 95% ethanol wash 2 times, the resulting precipitation with 1mL distilled water dissolved, that is, DNP-fish essence protein solution, spare.
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