Serum lipoprotein agarose gel electrophoresis.

related topics . Experimental Objective: To furtherthe basic principles of electrophoresis and the principles of gel electrophoresis..2. Familiar with
characteristics and
of agar gel electrophoresis of agar gel..3. Master the classification of plasma lipoproteins in normal people..Principle of agarose gel electrophoresis: Agarose gel is a straight chain polysaccharide consisting of D-semi-lactose and 3,6 dehydrated L-semi-lactose residues alternately arranged by hydrogen bonds. Electrophoresis, because the gel contains a large amount of water (98% to 99%), solid support less impact, so electrophoresis speed, zone neat. And because agar sugar does not contain charged groups, electrolysis effect is very small, is a good electrophoresis material, the separation effect is better.. Serum
lipids are combined with lipoproteins to form water-soluble lipoproteins. The types and quantities of lipoproteins contained in various lipoproteins vary greatly from lipoprotein particle size. Therefore, agar glycogel as a support, in the electric field can make a variety of lipoprotein particles separate..The method of electrophoresis separation of agarose gel serum lipoprotein is simple. Pre-dye serum lipoprotein with lipid dye sultan black (or oil red, etc.). The pre-dyed serum is then placed on agarose gel plate for separation.
after powering on, it can be seen that lipoproteins are divided into three zones, from negative to positive in order of β lipoproteins (deepest), front β lipoproteins (the shallowest) and α lipoproteins (slightly deeper than the previous β lipoproteins), at the origin should be free of milky particles. This method is applied to the type of hyperliproteinemia, can be divided into several different types of serum lipoprotein, for different types of hyperlipidemia, coronary heart disease, hypertension and myocardial infarction clinical diagnosis to provide
bio-chemical
indicators.. Equipment and
reagentsequipment electrophoresis, electrophoresis tank,
centrifuge
, water bath pot, dyeing tray, micro-injector, filter paper, tweezers and scissors, 2cm/> ́8cm/> glass tablets 2.reagents (1) barbie buffer (pH8.). 6): Said to take barbito sodium 15.4g/>, barbito 2.76g/> and
EDTA
0.29g/>, after water dissolution, then add distilled water to 1,000ml (pH is 8.6, ion strength 0.075), as electrode buffer..(2) Sudan black dyeing liquid: sultan black 0.5g/> dissolved in 5 ml of waterless ethanol to saturation.. (3) Gel buffer: said to take trihydroxymethane (Tris) 1.212g/>, EDTA0.29g/> and NaCl5.85g/>, dissolved with distilled water, diluted to 1,000ml, pH to 8.6. . (4) agarose gel: said to take agar sugar 0.50g/> dissolved in 50 ml gel buffer, add water 50 ml, in the water bath
heat
to boil, until agar sugar completely dissolved, immediately stop heating. . (5) Fresh blood (insoluble blood phenomenon). . Operation steps: 1. Pre-dyed serum serum 0.2 ml plus sultan black dye 0.02 ml in
test tube
, mixed rear 37 degrees C/> water bath dyed 30min, then centrifuged (2,000r/min) about 5min. . 2. Preparation of agarose gel board 0.5% agarose gel prepared in a boiling water bath (or microwave oven) heating and melting, with a 10 ml straw to absorb about 3 ml of gel solution poured on the slide, after about half an hour of solidification (day heat needs to be extended, can be placed in the refrigerator for a few minutes to accelerate solidification). . 3. Dot pre-dyed serum At 2cm/> from one end of the solidified agarose gel plate, remove the gel vertically with a homemade puncher (fixing two small pieces of film on both sides of the small glass sheet) and then peel out the small strip of gel with the film. Use a small piece of filter paper to absorb moisture from the small groove, take care not to damage the gel on the edge of the groove. Finally, a trace syringe is used to absorb the pre-dyed serum about 20m/>l into the small groove on the gel plate. . 4. Electrophoresis places the serum-added gel plate parallel to the electrophoresis tank and the sample at the negative end. Wet with two layers of filter paper or gauze in barbito buffer, and then gently closely attached to both ends of the gel plate, the other end of the gauze immersed in the electrophoresis tank Barbie buffer, power supply, voltage of 100 to 120V, each current of 3 to 4mA, about electrophoresis 40 to 60min, visible separation lipoprotein ribbon. . Note: 1. Pre-dyed serum is related to temperature, low temperature coloring is slow, high temperature coloring is fast, 37 degrees C/> is more suitable. . 2. Pour agarose gel plate to make as much thickness as possible, otherwise it will affect the separation effect of lipoprotein. . 3. Put the gel plate into the electrophoresis tank, should remember that parallel with the power line, the sample end negative pole, bridge filter paper can not be placed on the sample. . Question: What are points of the agarose gel electrophoresis? . 2. Why can't normal people see celiac particle belt when serum lipoprotein electrophoresis? . 3. What are the precautions for agarose gel electrophoresis?
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