EXPERIMENT 5 POLYACRYLAMIDE GEL DISC
ELECTROPHORESIS OF SERUM LIPOPROTEIN
PRINCIPLE
Dye the serum lipoprotein with the Sudan black B propanediol in advance, and
add it in the polyacrylamide gel electrophoresis（PAGE） column to migrate. Because
of molecular sieving effect, PAGE has high resolution and can separate every
constituent of the serum lipoprotein clearly.
PROCEDURE
1. Gel preparation: Insert several 0.5×7.5 cm clean glass tubes on the rubber
seat perpendicularly to prepare gel. Take out the following reagents from the
refrigerator. Balance in the room temperature and then add them in a small triangle
bottle one by one:
20% monomer solution
2.0ml
Tris—EDTA-Na2 buffer
1.2ml
Distilled water
6.6 ml
TEMED(tetramethyl ethylenediamine)
0.01ml
10% ammonium persulfate solution
0.15ml
(After adding in ammonium persulfate and mix lightly, the reagents begin to
polymerize. Therefore, the column filling should be finished within 5 minutes.)
2. The column filling: Draw the above-mentioned mixture rapidly by a dropper
and inject it into the glass tube along the tube wall, each tube 0.8～1.0ml. Then, add
in distilled water slowly by a dropper along the tube wall, approximately 0.5cm high,
to isolate the oxygen. Put the column in room temperature for 30 minutes. The clear
interface appearing between the gel surface and the water indicates the gel
polymerization has completed. Pour away the water and absorb the residual water on
the gel surface gently with filter paper strips. Pay attention not to damage the gel
surface that has already polymerized.
3. Sample preparation and adding: Take 0.2ml serum and add in 0.02ml Sudan
black B propanediol solution and 0.02ml 25% sucrose solution. Mix them up. Settle
the mixure at room temperature for 45 minutes. Centrifuge it 5 min at 2000 rpm.
Take 30μl dyed serum. Add it on the gel surface and then top up electrophoresis
buffer slowly along the tube wall.
4. Electrophoresis: Take down the tube from the rubber seat and insert it in the
hole of the electrophoresis chamber tightly to avoid leaking. The tube must be
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vertical. Fill electrophoresis buffer into the upper and lower chambers separately. The
upper chamber connects the cathode and the lower chamber connects the anode. Let
electric current be 2 ~ 5 mA per tube. When the first band is about 1cm away from
the tube bottom, the electrophoresis can be stopped. It takes about 20 ~ 30 minutes.
5. Gel peeling: Pour the upper chamber buffer (put in another bottle), and take
down the tube. Take a 2ml syringe with a long needle (about 10cm), and fill it with
distilled water. Between the tube wall and the gel, insert the needle along the wall
carefully, rotate the tube and inject the distilled water at the same time. With the
pressure and lubrication of water, the gel column leaves the tube. If necessary, after
the needle peeling, the gel column can be blown out slowly by an ear wadhing bulb.
6. Observation and drawing: Observe the shade of colour, width and order of
the bands and record them.
QUESTIONS
1. Which bands may appear in the electrophoresis of normal limosis serum
lipoprotein? Compare this electrophoretogram with that of agarose electrophoresis of
serum lipoprotein and explain the difference.
2. Is each lipoprotein band separated into uniform by electrophoresis? Why?
REAGENTS
[1] Tetramethyl ethylenediamine (TEMED)
[2] 10% ammonium persulfate solution: Make 0.5 g ammonium persulfate
dissolved in some distilled water and add in distilled water again to 5 ml. Preserve in
the refrigerator and the storage time can't exceed one week. It’s better to prepare
before using.
[3] 25% sucrose solution: Make 12.5 g sucrose dissolved in 50 ml distilled
water.
[4] 0.5mol/L Tris - 0.04mol/L EDTA - Na2 buffer:
Dissolve and dilute 6.057 g Tris(hydroxymethyl)aminomethane and 1.17 g
EDTA - Na2 to 100 ml with distilled water, modulate pH to be 8.8. Place the solution
in the brown bottle and keep it in the refrigerator.
[5] Take 19.6g acrylamide (Acr) and 0.4 g bis-acrylamide (Bis), and dilute to
100 ml after dissolve them with distilled water. (Dissolve Bis first and then add in
Acr. After fixing the capacity, filtrate the solution to remove the insoluble matters.)
[6] The electrophoresis buffer: One may select any kind of buffers below.
(1) 0.025 mol/L boric acid—borax buffer: pH 9.0.
Take 90ml 0.4 mol/L boric acid—borax buffer, and add distilled water to
1440ml. This buffer is quite suitable.
The method to prepare 0.4 mol/L boric acid—borax buffer: Take 30.512 g
borax and 4.948 g boric acid, dissolve them in distilled water, and dilute them to
2
1000 ml.
(2) The barbital buffer: pH8.6 I=0.075.
Take 15.458 g barbital sodium and 2.768 g barbital, dissolve them in 300 ~
400m1 distilled water and add distilled water to 1000ml.
(3) Tris—glycine buffer:
Take 6.0g Tris and 28.8 g glycine, dissolve them in about 850 ml distilled
water, adjust pH to 8.3 with Tris or glycine, add distilled water again to 1000 ml and
keep it in the refrigerator. The cost of this buffer is high.
[7] Sudan black B propanediol solution: Heat up 20ml propanediol to
100~110℃, add in 0.2g sudan black B, stir solution for 5 minutes, and filtrate it with
No3 Xinhua filter paper while solution is hot. Keep this solution in the dark place
and it can stabilize at least for one month.
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