Serum protein cellulose acetate film electrophoresis.

related topics .. Objective: To master
basic principles
basic
electrophoresis
serum protein and electrophoresis.
2. Familiarize yourself with the meaning and clinical significance of the serum protein vinegar cellulose thin film electrophoresis map.

The basic principle of electrophoresis
The phenomenon of charged particles moving in an electric field in the opposite direction to its electricality is called electrophoresis.
different charged particles swim at different speeds in the same electric field during electrophoresis. Electrophoresis speed of charged particles (spherical
molecules
) in the electric field (
V
)As can be seen from the upper, the speed at which charged particles move in the electric field (
V
) and the particle belt The amount of charge (
Q
) and the strength of the electric field (
E
) are inversely inversely related to the size of the spherical molecule (with a radius of
r
) and the viscosity (η) of the medium in which it is located. Therefore, when electrophoresis is performed in the same electric field or medium, particles of different sizes can be separated by electrophoresis with different charges.
In practice, in order not to consider the impact of different voltages on electrophoresis speed, we often use the migration rate (move rate, M) to represent the electrophoresis characteristics of charged particles, the migration rate refers to the electrophoresis speed of charged particles at the strength of the unit electric field, i.e. . It can be seen that the more net charge of charged particles, the smaller the molecular particles, the faster the migration rate in the electric field; However, electrophoresis speed and electrophoresis migration rate are two different concepts, the latter is conducive to the comparison of electrophoresis results under different electric field strength, the migration rate measured by various charged particles under certain conditions is a constant.
2. The main factor affecting electrophoresis
(1) the pH of electrophoretic media: for both sex molecules such as protein and
amino acid
, the pH value of electrophoresis media affects the ionion of the protein, which determines the charge of the protein (
Q
). The pH value is less than the isoelectrelectration point, the molecule has a positive charge, swims to the negative pole, and if the pH is greater than the isoelectre, the molecule is negatively charged and swims to the positive pole. The farther away the pH value deviates from the isoelectre, the more net charge the molecule has, and the faster it swims. When the buffer pH is equal to its isoelectractive point, the molecule is in an isoelectrelectratic state and does not move. Since the isoelectrins of serum proteins are mostly between pH4 and 6, the separation of serum proteins is commonly used in pH 8.6 barbito buffers or trihydroxy methamethane (Tris) buffers.
(2) Ion strength of the buffer: low ion strength, fast electrophoresis, the separation zone is not easy to clear; If the ion strength is too low, the buffer buffer is small, it is not easy to maintain the constant pH, the ion strength is too high, then reduce the charge of the protein (compressed double layer) to slow down the electrophoresis speed. Therefore, the common ion strength is between 0.02 and 0.2.
(3) the intensity of the electric field: the intensity of the electric field is in a positive relationship with the speed of electrophoresis. The intensity of the electric field is calculated by the potential difference per centimeter, also known as the potential gradient. If the filter length of the paper electrophoresis is 15cm and the voltage (potential difference) at both ends is 150V, the electric field strength is 150 / 15 x 10 V / cm, and the higher the electric field strength, the faster the charged particles move. With the increase of electric field strength, the current intensity increases, and the heat production increases. The adverse effects of heat production are: (1) causing evaporation of water, changing the pH and ion strength of the solution, and (2) causing high media temperature, which can denature proteins. Therefore, electrophoresis must control the voltage within a certain range, when high-voltage electrophoresis, must be equipped with an effective cooling device.
(4) electro-penetration: In an electric field, the solution moves in a certain direction due to the fact that the ions in the water absorbed by the porous support make the surface of the support relatively charged, and under the action of the electric field, the solution moves in a certain direction, a phenomenon known as electro-penetration. For example, the filter paper cellulose used in electrophoresis on paper carries a negative charge, and in
agar
glycosit swimming, the agar sugar used is also negatively charged due to the presence of a large number of sulfates, which induces water to produce hydrohydrated hydrogen ions (H-3O). Under the effect of the external electric field, the water moves to the negative pole. If the measured sample is also positively charged, it moves faster, and if the measured sample is negatively charged, the movement slows down. Therefore, when electrophoresis, the speed of particle swimming is determined by the speed of swimming of the particles themselves and the electro-permeation of the solution. Therefore, when selecting support, we should try to avoid high electrical seepage of the substance.
3. Acetate cellulose film electrophoresis
cellulose acetate film electrophoresis is an electrophoresis technology that uses cellulose acetate film as a solid support. Acetic fiber film has a uniform foam-like structure (about 120 μ m thick), strong permeability, no resistance to molecular movement, use it as a regional electrophoresis support, less sample size, clear separation, no adsorption, wide application range and fast and simple, and dyed film can be soaked with ethanol and ice acetic acid solution transparent, transparent film easy to save and quantitative analysis.
experiment, cellulose acetate film as a support, at one end of the film with trace serum, the two ends of the membrane connected to the buffer. The isoelectre points of serum proteins are lower than pH7.0, electrophoresis often uses pH8.6 buffer, therefore, all kinds of proteins are negatively charged, in the electric field to the positive pole movement, because of different swimming speed is separated. Cellulose acetate thin-film electrophoresis separates serum proteins into clear proteins A, alpha1, alpha2, β and
γ
-globulin, and after dyeing proteins, they can be qualitatively observed by the location of the dyeing area band, or quantitatively measured by each color band.
equipment

. Equipment
elwimming meter
, cellulose acetate film (2.5 cm ×8cm), dyeing liquid plate, rinse plate, tweezers
2. Reagent
(1) Barbito buffer (pH8.6, ion strength 0.075): Barbito sodium 15.46g, barbito 2.78g plus distilled water hundreds of milliliters,
heating
dissolved and then added distilled water to 1000ml, mixed.
(2) dye: amino black 10B 0.5g, dissolved in methanol 50ml, add ice acetic acid 10ml, distilled water 40ml mixed.
(3) rinse: methanol (or 95% ethanol) 45ml, ice acetic acid 5ml, distilled water 50ml mixed.
(4) sequestration: 0.4mol/L NaOH solution
(5) clear solution: 95% ethanol 75ml, ice acetic acid 25ml mixed.

Operating Step
) 1. Dot the acetic acid fiber film (2.5cm×8cm) one, immersed in barbiture buffer, to be fully soaked, remove gently clamped in the filter paper, absorb excess solution, and then the film's lightless At one end of the glossy surface 2.0cm, use a small slide to extract a little serum, printed vertically on the membrane, until the serum seeps into the film, with a non-glossy face down, at both ends with several layers of soaked filter paper (or gauze) fastening, covering, balancing 2 to 3min, and then powering on.
2. Power-on General voltage is 110 to 140V, current is about 0.4 to 0.6mA / cm, and time is 45 to 60min.
3. Immediately after dyeing off the power supply, remove the membrane, put it in the dyeing fluid to soak 2 to 3min, and then move into the rinse lotion to rinse several times, until the protein strip is clear and the background is colorless.
4. Quantified test tube 6, numbered 0 (blank), A, alpha1, alpha2, β,
γ
, electrophoresis map is also according to A, alpha1, alpha2, β,
γ
protein zone belt cut open, respectively, loaded into the corresponding number test tube. Then cut a blank strip about the width of the alpha1 band at both ends of the map and place it in a blank tube. Add 0.4mol/L NaOH 4ml to each tube, shake several times, so that the dye color leaching, after 30min, at 620nm wavelength color, with blank tube correction zero point, read the clear protein and alpha 1, alpha2, β and
γ
globulin tube absorption.

the sum of the absorbents T is the sum of the various protein absorbents:
T

A
, alpha1 , alpha2 , β ,
γ
. The percentage of proteins for each group is calculated according to the following calculation:
protein (%) 100%

1 globulin (%) × (α A α/
T
) ( α 1 /
T
) × 100%
α 2 globulin (%) ( α 2 /
T
) × 100% β globulin (%) ( β /
T
) × 100%
γ
globulin (%) (
γ
/
T
) × 100%
many laboratories now use photodrometer quantification. After the film
completely
dry, immerse it in a clear liquid of about 5 to 10min, remove the flat paste on the glass plate, and dry completely to become a transparent film. The absorbent density is then measured on the light density meter and can be plotted into curves, or the percentage content of various proteins can be calculated directly.

Clinical Significance 1. Reference Value
Serum Protein Reference Value Isoelectrelation Point Molecular Weight percentage of
Total Protein
). Clear protein alpha1 globulin alpha2 globulin β globulinγ
globulin 4.64 5.06 5.06 5.12 6.85 to 7.3 69 000 200 000 300 000 90. 000-150 000 156 000-950 000 57-72 2-5 4-9 6.5 to 12 12-20 2. Clinical significance Serum protein acetate fiber membrane electrophoresis is often used in clinical analysis of blood, urine and other samples of proteins for clinical diagnosis of liver, kidney and other diseases reference. For example, in patients with nephropathy syndrome, the albumin of small molecular weight of plasma protein leaks out of the body with urine, resulting in significantly smaller and shallower albumin regions in the electrophoresis map of cellulose acetate film. For example, in patients with chronic hepatitis and cirrhosis, the liver's ability to synthesize plasma proteins is greatly reduced due to damage to liver cells, resulting in a significant decrease in plasma albumin and a relatively significant increase in γ globulin. Abnormal globulin bands are visible in the electrophoresis map of serum protein cellulose acetate film in patients with multiple myeloma.
note is not
. 1. Electrophoresis, the voltage should be controlled at 110 to 140V, can not be too high, if the voltage is too high, the production of heat, then protein specimens will be destroyed, and the voltage is high charged particles move fast, the separation effect is not ideal.
2. Cellulose acetate film before electrophoresis, be sure to soak in the buffer, otherwise it will prevent electrophoresis separation, preferably soaked overnight, the best effect.
3. Samples must be pointed at a glossy surface, otherwise it is difficult to inhale, the sample size should not be too much (serum samples are most suitable for 3 sl). The reason is that cellulose acetate film has limited protein tolerance. Too much amount of material with similar molecular weight competes with each other for migration and overlaps, affecting the observation of results.
4. After electrophoresis begins, the film can no longer be taken and released to prevent electric shock. If necessary.