Glucosamine gel layering.

1.
the properties of glucosaccharin gel and the principle of gel layering.
2. Learn the basic operating techniques of glucosaccharin gel analysis.Gel layering, also known as molecular blocking layering or gel
filtration
, is a layer separation technique based on the molecular weight differences of the separated substance, which provides a very gentle separation method for purifying biological large molecules such as
proteins
and so on. The fixed phase carrier of layering is the gel particle, which is widely used at present: Sephadex and
agar
sugar gel (Sepharose) with various aperture ranges.
glucosaccharide gel is a polymer compound with porous mesh structure formed by the cross-linking of glucosaccharide molecules and crosslinkers 3-chlorine 1,2-epoxy propane. The size of the mesh holes in the gel particles can be controlled by adjusting the ratio of glucosaccharides and crosslinkers, the greater the cross-linking degree, the closer the mesh structure; The sethable molecular weight ranges from a few hundred thousand to hundreds of thousands.
Glucosamine gel layering, is to make the substance to be separated through the glucosaccharin gel column, each component due to different molecular weight, in the gel column by different blocking effect, and in the analysis column at different speeds. Substances with a molecular weight greater than allowed into the gel mesh hole range are completely blocked by the gel, can not enter the inside of the gel particles, the blocking effect is small, with the solvent flowing between the gel particles, so the process is short, and the first flow out of the column; If the molecular weight of the separated substance is between the molecular weight of the fully blocked and the fully entered mesh material, it flows out of the column between the two, thus achieving the purpose of separation.
experiment used G-25 as a fixed phase carrier to separate blue glucosaccharide-2000 and bromophenol blue. Blue glucosaccharide - 2000 molecular weight close to 2×106, while bromophenol blue molecular weight of 670, the molecular weight of the two is quite different, the former is completely blocked, and the latter can fully enter the gel particle mesh hole, the two through the time of the column is different. (Experimental Materials)
1. Experimental equipment
layering column (1×
20cm) with a small section of latex tube and spiral clip
; The
2. Experimental
Reagent (1) Tris-Acetic Acid Buffer (pH7.0): Take 0.0lmol/L Tris solution (containing 0.1mol/L KCl) 900ml, pH to 7.0 with thick acetic acid, and distilled water to 1000m1.
(2) Bromophenol Blue Solution: 10 mg of Bromphenol Blue, dissolved in 5 ml of ethanol, stirred thoroughly to dissolve it, and then added tris-acetic acid buffer (pH7.0) one by one until the solution is dark blue.
(3) blue glucosaccharide - 2000 solution: called blue glucosaccharide - 2000 10 mg, dissolved in 2 ml Tris-acetic acid buffer (pH7.0).
(4) sample solution: take bromine phenol blue solution 0.1 ml, blue glucosaccharide - 2000 solution 0.5 ml mixed for the upper column sample solution.
(5) G-25 (Sephadex-G-25) . Experimental operation:
1. Preparation of experimental gel: commodity gel is
driding
particles, when used by swelling treatment, said to take 4 grams of glucosaccharide gel G-25, plus 50 ml distilled water, stirred well, dissolved at room temperature for 6 hours, or boiling water bath swelling for 2 hours, generally using the latter method. Then use pouring method to remove the gel upper water and fine particles, with distilled water repeatedly washed several times, and then to buffer solution (pH7.0 Tris-acetic acid solution) wash 2-3 times, so that pH and ion strength to achieve balance, and finally remove the solution and gel particles internal bubbles, gel can be stored in the buffer.
2. Mounting column: wash the triad column, fixed vertically on the iron
stent
, select the film end as the lower port of the column, connect the lower port to the latex tube and clamp it with a spiral clip. Add sequestration to the layer column, open the lower helix clip, let the solution flow out, remove the residual bubbles, and finally retain about 2 cm height of the scavenging fluid, tighten the screw clip. Gently stir the gel evenly, with a glass rod along the inner wall of the column slowly injected into the column, until the gel deposited under the column bed has exceeded l cm, open the lower helix clip, continue to load the column to the column bed height of 8 cm, close the exit. Bubbles are strictly prohibited during the loading process, as far as possible once installed, to avoid layering. Then balance the volume of l to 2 column beds with semen, and always maintain a certain amount of semen on the gel surface. After balancing, tighten the lower screw clip.
3. Add sample: Open the screw clip to allow the escape fluid on the cylinder surface to flow out until the bed surface is just level with the liquid surface, close the lower outlet. Take bromophenol blue and blue glucosamine - 2000 mixture 0.3 ml, carefully added to the gel surface, do not stir the surface of the column bed. Open the lower outlet so that the sample solution enters the gel and begins to collect the fluid. Close the lower outlet when the sample solution is flowing exactly to level with the gel surface. Wash the analysis column sample area with a small amount of semen, wash three times, each cleaning liquid should be fully into the gel column, and then carry out the next washing. Finally, the semen is added to the gel surface to maintain a height of 3-4cm.
4. Debaulation and collection: connect the gel column layering system, adjust the flow rate of the wash-out liquid at 1 ml per minute, to be washed away. Careful observation of the separation of samples in the analysis column, collection of elesesion, each collection of 3 ml is replaced by a collection tube (test tube pre-numbered), collection of about 20 tubes, the sample can be completely elused. The semen in each collection tube was measured at a wavelength of 540nm using a 721-type hydrometer.
5. Gel recovery treatment method: after completely washing off the sample, continue to use three times the size of the column bed washing gel, the lower mouth of the column in a small
spower
, slowly open, and then slowly release the upper mouth, so that the gel is all recycled into a small berrock, spare.results,
the curve is to take the de-pipe number as the horizontal coordinates and the light density as the ordinate as the ordinate. Analyze the graph and discuss the results of the experiment. . Why
different samples can be separated by glucosaccharin gel analysis?
2. What problems should I pay attention to when analyzing glucosaccharin gel? .