Experimental method for separation of serum proteins by cellulose acetate membrane electrophoresis
Experimental principle
A charged protein, which migrates in an electric field toward an electrode that is electrically opposite to its charge, is called electrophoresis. The isoelectric points of various proteins in serum are different, but most of them are below pH 7. If the serum is placed in a buffer of pH 8.6, these proteins are all negatively charged and move to the anode in the electric field. Since various proteins have different negative charges and different molecular sizes in the same pH environment, the speed of migration to the anode in the electric field is also different. Protein molecules are small and charged, and the migration speed is fast; otherwise, the swimming speed is slow. Therefore, the serum protein can be divided into five regions of albumin, a1-globulin, a2-globulin, b-globulin and g-globulin, and the percentage of serum protein content can be calculated by staining.
Acetate fiber membrane (CAM) is used as a supporting medium, and after electrophoresis separation, it can be dyed to display a clear protein electropherogram. Since the binding of dye to protein is proportional to the amount of protein during dyeing, each protein band is cut and eluted with a certain amount of NaOH diluted solution to perform colorimetric determination of the relative content of each protein band. . The CAM can also be dissolved into a transparent film by using a certain amount of an organic solvent and subjected to scanning quantification using a densitometer.
Acetate fiber membrane electrophoresis has the advantages of micro, rapid, simple and high resolution, and is widely used in the separation and determination of serum proteins, hemoglobin, lipoprotein and isoenzyme.
Experimental reagent
1. Barbital-Barbital sodium buffer (pH 8.6, m=0.06). Weigh 2.21 g of barbital and 12.36 g of barbital sodium, dissolve in 500 ml of distilled water, and dissolve by heating. After cooling to room temperature, dilute to 1000 ml with distilled water.
2. Staining solution
(1) Lichunhong S staining solution: Weigh Lichun red S O.4g and trichloroacetic acid 6g, dissolve with distilled water and dilute to 100ml.
(2) Amino black 10B staining solution: 0.1 g of amino black (C22H13O12N6S3Na3) 10B was weighed and dissolved in 20 ml of absolute ethanol, and 5 ml of glacial acetic acid was added to dissolve it. Another 2.5 g of sulfosalicylic acid was dissolved in a small amount of distilled water, and the two liquids were mixed and shaken by adding the pre-liquid, and then made up to 100 ml with distilled water. Weigh Lixihong S 0.4 g and 6 g of trichloroacetic acid; dissolve in distilled water and dilute to 100 ml.
3. Rinsing solution.
(1) 3% (V/V) acetic acid solution: suitable for rinsing of Ponceau S staining.
(2) Mix 45ml of methanol, 5ml of glacial acetic acid and 50ml of distilled water, which is suitable for rinsing of amino black 10B dyeing.
4. Transparent liquid Take 75 ml of absolute ethanol and 25 ml of glacial acetic acid and mix for use.
5. Eluent
(1) 0.1 mol/L NaOH solution: used for elution of Ponceau S staining.
(2) 0.4 mol/L NaOH solution: for elution of amino black 10B staining.
6.40% (V/V) acetic acid solution.
Experimental equipment electrophoresis apparatus, electrophoresis tank, spectrophotometer or densitometer.
Experimental materials Acetate film, petri dish, filter paper, tweezers, spotter, ruler, pencil, scissors, etc.
Experimental procedure
1. Preparation of the electrophoresis tank
Add barbiturate-barbital sodium buffer to the electrophoresis tank (inject the same amount of buffer into the groove on both sides), adjust the buffer on both sides to be in the same horizontal plane, and the distance between the liquid surface and the support is about 2 to 2.5 cm. The width of the bracket is just right for the length of the CAM, and is bridged with 3 to 4 layers of filter paper or 4 layers of gauze.
2, CAM preparation
Take vinegar fiber film (2 cm * 8 cm) on the matte side of CAM (coated with cellulose acetate) 1.5 cm from one end with a ruler and a pencil to draw a line (perpendicular to the long axis of CAM), as a spot mark After the film strip is numbered, immerse the matte side down into the barbiturate-barbital sodium buffer solution, and take it out after fully saturating (generally about 20-30 min), clamp it in a clean filter paper, and remove excess Buffer.
3, spotting
Use a serum sampler to uniformly position 3-5 ul of non-hemolyzed fresh serum at the scribe line. The sample should be kept at a distance from the edge of the membrane to avoid deformation of the protein band in the electropherogram. After the serum penetrates into the membrane, the spotter is removed. It should be noted that the amount should be appropriate, uniform and vertical, and avoid breaking the film.
4, balance
Place the sampled film face down, place the sample end on the cathode end, attach the film strip to the salt bridge of the electrophoresis tank holder and keep it straight, and the other end of the bridge hangs into the buffer, the salt bridge Both ends of the membrane were connected to the buffer, and the tank lid was equilibrated for 5 min, and then electrophoresis was performed.
5, electrophoresis
Connect the positive and negative electrodes corresponding to the electrophoresis tank and the rectifier correctly. Connect the anode to the side and the anode to the other side. Turn the power on. Adjust the voltage of 10V ~ 15V / cm film length, current 0.4 ~ 0.6mA / cm total film width, electrophoresis 40-60 minutes (usually 45min in summer, 60min in winter), when the electrophoresis zone is unfolded 3.5cm ~ 4cm can be closed power supply.
6, dyeing
After the power is turned on, the film is immediately taken out and directly immersed in the Ponceau S or the amino black 10B staining solution, and dyed for 5 to 10 minutes.
7, rinse
Prepare at least 3 to 4 rinse dishes and load the rinse solution. The film strips were removed from the staining solution and the staining solution was drained as much as possible, and the rinses were repeatedly rinsed in sequence until the background was bleached. Five ribbons are clearly visible at this time. Waiting to be done.
8, quantitative
(1) Elution colorimetric method Six test tubes were taken to indicate "A, α1, α2, β, γ, blank". The rinsing film was cut into the respective tubes, and an average size film strip was cut from the blank background and placed in a blank tube, and eluted according to the following method depending on the staining.
1 amino black 10B staining and elution: 6 tubes were added with 6 ml of 0.4 mmol/L NaOH solution in the albumin tube, and 3 ml of each of the other 5 tubes were added, shaken several times, and placed in a 37 ° C water bath for 20 minutes to completely leaching the color. With a wavelength of 620 nm, the tube was zeroed with a blank tube, and the absorbance of each tube was read, wherein the absorbance of the albumin tube was ×2.
2 Lichunhong S staining elution: The eluent was diluted with 0.1 mmol/L sodium hydroxide solution, and the amount was the same as above. After 10 minutes, 0.6 ml of 40% (v/v) acetic acid was added to the albumin tube, and 0.3 ml of each of the remaining 5 tubes was added to neutralize part of the sodium hydroxide to make the solution darker. If precipitation occurs, the supernatant color can be centrifuged. The wavelength was adjusted by a blank tube at a wavelength of 520 nm, and the absorbance of each tube was read, wherein the absorbance of the albumin tube was ×2.
(2) Optical density scanning method 1 Transparent: immerse the CAM strip that has been rinsed and dried in a transparent liquid for 3 to 5 minutes, remove it and lay it on a clean and dry glass slide (no air bubbles), and remove too much transparent liquid in an upright moment. . Bake in an oven at 90-100 ° C for 5-10 minutes, remove and cool to room temperature. This method is transparent CAM, the protein area is distinct, the film is flat, and can be directly scanned or permanently preserved. If the ten naphthyl hydrogen or the liquid stone vinegar is transparent, the rinsed film should be dried and then transparent, and the permeable film can not be stored for a long time, and wrinkles are likely to occur.
2 Scanning quantification: Put the transparent film into the optical path of fully automatic densitometer or other optical density scanning, select the wavelength of 520nm, trace the peaks of each protein zone, and calculate the relative content of each protein component.
9, calculation
In the quantitative calculation, first calculate the sum of the respective optical density values: recalculate the percentage of the protein in each part of the serum. The sum of absorbance (T) = 2A + α1 + α2 + β + γ (absorbance)
Relative percentage of serum protein components = Ax / AT × 100%
Ax represents the absorbance of each globulin component (2A + α1 + α2 + β + γ).
A%=2A/ T×100% β=β/ T×100%
11%=α1/ T×100% γ%=γ/ T×100% α2%=α2/ T×100%
Protein content of each component (g/L) = (% of each component protein (%) × total serum protein g / L) / 100
10. Clinical significance (1) The normal reference value of serum protein vinegar membrane electrophoresis is:
Protein component g/L as a percentage of total protein (%)
Albumin 35～52 57.0～68.0
11 globulin 1.0～4.0 1.0～5.7
22 globulin 4.0～8.0 4.9～11.2
Beta globulin 5.0～10.0 7.0～13.0
γ globulin 6.0～13.0 9.8～18.2
(2) Clinical significance Electropherogram can provide a basis for clinical disease diagnosis.
Nephropathy can be seen in acute and chronic nephritis, nephrotic syndrome, renal failure, etc., the pattern shows Alb decreased, α2 and β increased; cirrhosis type can be seen in chronic active hepatitis, cirrhosis, etc., the pattern is Alb Decrease, β and γ increase, there may be a “β-γ†bridge in which β and γ are difficult to separate and connect together. This phenomenon is caused by the increase of IgA caused by liver fibrosis; the phase type of acute reaction is often increased by α1 and α2. It is characterized by chronic inflammation, Alb is decreased, α2, γ is more common; M proteinemia is mainly seen in multiple myeloma, patients have a large number of monoclonal proteins (mainly IgG or IgA), and can be used in β and βγ A narrow band-shaped zone appears between them, called the M zone.
Precautions
1. Do not touch the buffer or CAM in the tank when power is on to prevent electric shock.
2. The electrodes should be exchanged each time the electrophoresis is performed so that the positive and negative ions of the buffer in the electrophoresis tanks on both sides are exchanged to maintain the pH of the buffer at a certain level.
3. The liquid level of the electrophoresis buffer should be kept at a certain height. If the pH is too low, the electroosmosis of gamma globulin may occur (gamma globulin moves to the cathode), and the liquid level on both sides of the electrophoresis tank should be kept at the same level. Otherwise, pass There is a siphon phenomenon in the film, which will affect the migration speed of protein molecules.
4. Common reasons for electrophoresis failure or unsatisfactory mapping (1) Electrophoresis map is not neat or protein components are poorly divided: too many spots; uneven spots, irregularities; film too wet, sample diffusion; spotting speed is too slow The surface of the film is partially dried or the film is not completely saturated or the temperature is too high, so that the film surface is partially dried or the water is evaporated; the buffer is deteriorated; the film is not placed properly during electrophoresis, skewed, bent, and is not parallel with the current direction; the sample is not fresh; the current is too low The quality of CAM is not good, the structure of the film is too fine, the water permeability is poor, and the conductivity is poor.
(2) The white coloration of albumin after dyeing is shallow: the staining time is not enough or the staining solution is old; the albumin content is too high, which can reduce the serum dosage or prolong the dyeing time.
(3) The electrophoresis speed is slow: the current is too low; the buffer supplied to the film is insufficient, the filter paper or gauze connecting the film and the buffer is too thin; the temperature is too low; the film structure is excessively fine, the water permeability is poor, the conductivity is poor; the buffer water evaporates, This causes the ionic strength to increase.
(4) The film is not transparent: the transparent liquid is old; the soaking time is insufficient; the film is placed at an oven temperature of less than 90 °C.
5. Dye problems: Various dyes have different affinities for serum components, and most dyes have greater affinity for albumin than globulin. For example, the binding strength of amino black 10B to globulin is 80% of albumin, so it often leads to high albumin results and low globulin. Dyeing with Ponceau S, the effect is better than that of amino black 10B. Lihong is an indicator that is red when PH<10 and purple when PH>12.5. Its maximum absorption peak in an acidic solution is about 523 nm, showing symmetry. In the normal concentration range of serum protein, Lichunhong S can be directly proportional to each protein component, while amino black 10B stains albumin too deeply, and it is easy to appear small spots in the zone, which is not ideal.
6. The sample is required to be on a rough surface (matte side), otherwise the sample is difficult to inhale into the film. When electrophoresis, it is best to place the side of the sample with the side facing down to prevent evaporation of water during electrophoresis and affect the electrophoresis results.
7. Specimens should be fresh and must not be hemolyzed. Hemolysis specimens cause a false increase in beta globulin, as hemoglobin electrophoresis is located within the beta globulin region.
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