Utilizing the differences in the number of charges and particle sizes of various apolipoproteins at a certain pH, so as to obtain different migration shelters in the electric field, to achieve the purpose of separation and determination of the experiment from Mudanjiang Medical College, undergraduate 5-year laboratory guidance for laboratory tests.
Operation method
Polyacrylamide gel discontinuous disk electrophoresis experiments
Principle
Utilizing the differences in the number of charges and particle sizes of various apolipoproteins at a certain pH, and thus obtaining different migration shelters in the electric field, to achieve the purpose of separation and determination.
Materials and Instruments
Kaumas blue Bromophenol blue aqueous solution Glacial acetic acid Move I. Experimental reagents: For more product details, please visit Aladdin Scientific website.
1. Gel stock solution preparation and working mix ratio table
2. Decolorizing agent: 1000 ml of ethanol, 320 ml of glacial acetic acid and water to 4000 ml.
3. Staining agent: komas blue 0.46g, dissolved in 400ml decolorant.
4. bromophenol blue aqueous solution: bromophenol blue 0.05g, dissolved in water to 100ml.
Second, the experimental operation
1. according to the above table given the proportion of preparation of separation gel, No. 3 reagent to be the last to add, and then quickly mixed. Immediately to the bottom of the glass tube has been sealed in the addition of the above separation of glue to about 2 / 3 of the length of the tube, and then gently along the wall of the tube to add a thin layer of water to close the air. Generally at 20 ℃ room temperature in about 40 minutes to form a gel. Pour off the water layer, inverted on filter paper to drain the water in the tube, and then according to the above table has been made of concentrated gel, No. 6 reagent last to join, mix quickly, immediately added to the separation of gel on top of the upper edge of the tube from the mouth of the 8mm, and then gently add a thin layer of water on top of the tube to make it polymerize under fluorescent light irradiation, about 20 minutes or so can be polymerization is complete, and pour off the water layer.
2. Open the bottom of the tube, insert the glass tube into the hole of electrophoresis tank (the separation gel is underneath), and the surrounding area should not be leaked.
3. Sample addition: first dilute the No. 4 solution (take 2ml of No. 4 solution and add water to 6m1). Take 0.5m1 of serum, add 1.5ml of No. 4 liquid release, add 3 drops of No. 7 and 3 drops of bromophenol blue reagent, mix well. Check whether the upper and lower ports of the gel tube have been immersed in the buffer, otherwise adjust. Pipette 30-35ul of the sample prepared above (about 7.5ul of serum) with a microfuge, and slowly extend the tip of the spiker along the wall of the tube to the gel surface at the upper end. Slowly add the sample to the gel surface (to form a layer and not scattered).
4. Electrophoresis: connect the upper tank to the negative pole, connect the lower tank to the positive pole, cover the electrophoresis tank, open the electrophoresis instrument switch to adjust the output voltage. Make each tube current flow 1-2mA, to be bromophenol blue indicator into the separation of glue, increase the output voltage, so that the current per tube increased to 4-5mA, when the bromophenol blue indicator swimming to the next end of the tube 0.5cm from the end of the next to stop electrophoresis.
5. Strip the gel: remove the tube from the electrophoresis tank, use a long needle syringe to suck up the water, insert the needle into the tube wall slowly, and rotate the glass tube while inserting and injecting water into it, so as to separate the gel from the inner wall of the tube, and then let it come out and put it into the staining solution.
6. Fixation and staining: Let the peeled out gel column stained in the staining solution for 30 minutes, the gel was fixed by the state, and the proteins were stained with blue color into a number of disc-like zones. Strip off the floating color: the stained gel column gently transferred to a small test tube, add the decolorant soak, and change the wash several times, until the background is clear and the zones are clear.