Glucosamine gel layer desalination

related topics
. The purpose isLearn how gel analysis works and how to operate
Master the technique of
protein
desalination using glucosaccharin gel 2. Principle
Gel layering, also known as gel
filtration
or draining. The main device of gel filtration is a layered column filled with
medium and
.
1. The characteristics of the laminate medium
(1) in water is insoluble solid phase; (2) is a chemically inert substance;
(3) ion group content is low; (4) particle size and mesh uniform;
(5) mechanical strength is strong; (6) has a variety of a pores to choose from.
currently used more glucosal gels, polyacrylamide gels,
agar
sugar gels and their derivatives. In particular, glucosaccharin gel (commodity name Sephadex) is the most commonly used layering medium. It is composed of a high
moleculecompound with three-dimensional structure insoluble in water that is cross-linked by a certain average molecular weight of glucosaccharides and crosslinkers 1-chlorine-2, 3-epoxy propane ( )
. Adjusting the ratio of glucosaccharides and crosslinkers allows for gels of different mesh sizes and models. When the smaller the glucosaccharide molecular weight, the greater the amount of crosslinking agent, the greater the cross-link, the smaller the gel mesh, the smaller the water absorption, the smaller the G value. The G value represents ten times the amount of water absorbed (mL) per glial glue. Sephadex G-25, for example, should absorb 2.5mL/g of dry glue. Commonly used glucosaccharose gels are available in a variety of specifications, such as G-10, G-15, G-25, G-50, G-75, G-100, etc. The type chosen in the experiment should be determined according to the size of the molecules of the separated mixture and the purpose of the work (see Appendix V).
2. Separation Principle
When the mixture is added to the gel column and passes through the gel column with the washelot, substances of different molecular sizes are subjected to different blocking effects. Particles close to or greater than the mesh molecules, can not enter the mesh of the gel, under the action of gravity, they along with the solvent between the gel particles along a shorter flow down the flow, subject to small blocking action, moving fast, first out of the column (this phenomenon is called being blocked). The minimum molecular weight of the block is called the blocking limit of the specification gel, and the molecules smaller than the mesh can seep into the gel mesh, they are washed away from one mesh to another, layer by layer diffusion, blocking effect, long process, slow movement, and therefore out of the column. At the outlet of the analysis column, we used multiple
tub tubes
to collect the semen step by step to separate the components of the mixture from each other.
When we extract proteins from biological
tissues
, we often need to desalinate proteins, we can use a layering medium for glucosaccharide gel G-25 (or G-15, G-50), with appropriate sedatives for washing, after gel layering, you can separate large molecule proteins from small molecular salts.. Experimental materials and equipment 1.
protein solution containing ammonium sulfate
2. Equipment
layer analysis column: inner diameter × column height: 1.0cm×25cm
titration rack, screw clip: 1
tick test tube: 10mL×14
pipe: 1mL× 1
: 250mL×1 50mL×1
drop tube: 2
wash balls: 2
bottle washers, test tube racks, pipe pipe racks, glass rods: 1 4.
each Reagents

1. G-25 (or G-15, G-50)
soluble gel method: according to the volume of about 4g per layer column G-25 in
beagging
, with excessive distilled water dissolved in the boiling water bath for 2 hours or at room temperature for more than 6 hours. Remove the finely grated gel from the upper layer by pouring and repeat 3 to 4 times. Avoid stirring vigorously during operation and prevent damage to its cross-linking structure.
2. BaCl2 solution (1%)
3. Coomas Bright Blue G-250
said to take 0.1g Coomas Bright Blue G-250, first dissolved in 50mL95% ethanol, then added 85% phosphoric acid 100mL, and finally with distilled water to 1000mL. Store in a dark place.
4. (6mol/L)
5. The
different buffers should be selected according to the different properties of the purified protein.