3.3.3 in di kim tra mu protein ly trch 3.3.3.

.1 Chun b ha cht a) Ha cht gel Pha gel phn tch (separating gel) c nng 12%T Cho cc thnh phn sau theo th t vo ng Falcon 15 ml sch : 4 ml 30% Acrylamide/Bis (29:1), 2,5 ml Tris HCl 1,5 M pH 8,8, 3,4 ml nc ct 2 ln kh ion, 0,1 ml 10% SDS. Sau khi cho y cc thnh phn trn theo ng th tch vo ng Falcon. Tip tc thm vo ng Falcon 50 l ammonium persulfate 10%, 10 l TEMED v lc nh ng Falcon vi ln (trnh to bt kh), dng pipettte loi 100 1000 l bm t t dung dch gel vo khun, gel sao cho mc dung dch gel cao hn 7 cm (trnh to bt kh trong khun gel). Sau , nh nhng t mt lp nc ct ln trn lp gel mt gel c phng. Ch gel ng (khong 20 30 pht). Vi 10 ml dung dch gel c th c 2 gel 100 x 100 x 0,75 mm. Pha gel gom (Stacking gel) c nng 4%T Cho cc thnh phn sau theo th t vo ng Falcon 15 ml khc: 1,3 ml Acrylamide/Bis (29:1) 30%, 2,5 ml Tris HCl 0,5 M pH 6,8, 6,1 ml nc ct 2 ln kh ion, 0,1 ml SDS 10%. Sau khi gel phn tch trng ngng hon ton, ht nc bn trn. Tin hnh lp gel gom bng cch dng pipette loi 100 1000 l ht 2 ml dung dch gel gom vo becher 50 ml, thm vo dung dch 10 l ammonium persulfate 10%, 2 l TEMED. Dng pipette trn u v bm vo khun gel. ng thi a lc vo gel to cc ging mu (Lu : trnh to bt kh trong khun gel). Sau khi gel gom trng ngng hon ton, ta tin hnh tho lc ra khi khun gel cn thn trnh to bt kh trong cc ging (Lu : nu c bt kh trong ging ta dng pipette loi 100 1000 l bm kh mnh vo ging lm v bt kh). Tip theo ta lp da gel vo b phn in di, cho dung dch in di vo bn. b) Chun b dung dch np mu (sample buffer) v dung dch m in di (electrode buffer) Pha dch np mu gm: 3,55 ml nc ct 2 ln kh ion; 1,25 ml Tris-HCl 0,5 M pH 6,8; 2,5 ml glycerol; 2 ml SDS 10% (w/v); 0,2 ml bromophenol blue 0,5% (w/v). Khi dng thm vo 50 l 2- Mercaptoethanol hoc 0,5 ml dithiothreitol 2 M (DTT). Dung dch m in di l Tris glycine, pH 8,3. Pha 1l Tris glycine (196mM glycine, 0,1% SDS, 50 mM Tris HCl pH 8,3). c) Chun b dung dch nhum v dung dch gii nhum Dung dch nhum Coomassie Brilliant Blue R- 250 Pha hn hp theo t l nh sau vo mt hp nha c np: 0,2% Coomassie Brilliant Blue R- 250 (w/v), 45% methanol (v/v), 45% nc ct hai ln kh ion (v/v), 10% acid acetic (v/v). y kn li v gi trong ti n khi s dng. Dung dch gii nhum Pha hn hp sau theo ng t l vo mt hp nha khc: 25% methanol (v/v), 65% nc ct hai ln kh ion (v/v), 10% acid acetic (v/v). y kn, gi trong ti n khi s dng. 3.3.3.2 Chun b mu v chy in di Dng pipette loi 0,5 10 l ht 3 l dung dch np mu v 7 l mu protein vo mt eppendorf 0,2 ml sch, trn u, y np v gia nhit 950C t 3 5 pht gy bin tnh protein. Sau khi gia nhit c th ly tm 10.000 vng/pht trong 3 pht.

3.3.3.3 Tin hnh in di v xem kt qu 1. Dng pipette loi 0,5 10 l np mu vo cc ging (10 l/ging). 2. Tin hnh chy in di hiu in th 100 V, cng dng in 15 mA, trong thi gian 120 pht hoc in di cho ti khi vch mu ca dch np mu cch y gel khong 0,5 1cm. 3. Sau khi in di, tho gel ra khi khun, ra sch gel bng nc 5 pht (cn thn trnh lm t gel). Nhum gel trong thi gian 30 45 pht. 4. Sau khi nhum xong, ra gel vi nc 5 pht. Tin hnh gii nhum bng cch ngm gel trong dung dch gii nhum cho n khi nn gel tr nn trong sut khng mu. Sau khi gii nhum xong, protein c pht hin nh cc vch mu xanh lam trn nn gel trong sut. 3.3.4 Th nghim kho st chn iu kin in di iu kin in di nh hng ln n kt qu in di. Nu iu kin in di khng ph hp vi protein th rt c th s lm cc bng protein b cong, hoc ccprotein b bin tnh do s hot ng tr li ca cc enzyme. ng thi n cng cth lm cho cc bng protein khng phn tch r rng. Trong th nghim ny chngti tin hnh kho st iu kin in di cho ph hp vi protein ca l la nhmchn ra iu kin in di thch hp. B tr 3 th nghim khc nhau (bng 3.3) Bng 3.3 Th nghim kho st iu kin in di Th nghim Gel gom 1 200 V, 13 mA, 20 pht 2 100 V, 20 mA, 10 pht 3 100 V, 15 mA, 20 pht

Gel phn tch 200 V, 18 mA, 100 pht 100 pht100 V, 20 mA, 80 pht 100 V, 15 mA, 100 pht

Mu s dng trong th nghim ny l mu protein ly trch theo quy trnh tt nht trong 3 quy tnh kho st. Ch tiu theo di: hnh dng cc bng protein v s phn tch ca cc bng trn gel. 3.3.5 Kho st nng gel Nng gel nh hng rt ln n phn tch ca cc bng protein trn gel. V nu nng gel cng cao th kch thc l gel cng nh li v s cn tr s di chuyn ca ca cc protein c kch thc cng ln. Ni cch khc l vi mi mt nng gel th cho php phn tch protein mt khong trng lng phn t xc nh. thy c mt cch r rng cc bng protein ca la trn gel, cn kho st nng ca gel phn tch cho ph hp vi protein tng s c ly trch t l la. Phi la chn nng gel sao cho cc bng protein trn gel c phn tch r rng. Tin hnh th nghim bng cch cc ming gel c nng gel phn tch khc

nhau v th nghim trn cc mu protein. Nng ca gel gom c gi nguyn l 4% T, nng ca gel phn tch c thay i 12% T, 13% T, 14% T. Ta tin hnh chy in di cng iu kin vi hiu in th n nh 100 V, cng dng in 15 mA, thi gian in di l 120 pht, nhum gel trong 0,2% CBB(w/v) nhit phng 250C. Electrophoresis
Electrophoresis is defined as the movement of charged particles when placed into an electrical field of varying electrical potential. Separation of charged particles during electrophoresis occurs when different molecules move at different rates. Electrophoresis is a tool that is used by clinical laboratory scientists/medical technologists to separate molecules prior to molecule identification. For example, serum proteins, when placed in a buffer of pH 8.6, become negatively charged and migrate to an anode, while the buffer particles migrate to a cathode. Several forces affect the charged molecule during electrophoresis. The molecule will move in the direction of its opposite charge. Negatively charged molecules (anions) will move toward the positively charged electrode, the anode. Likewise, positively charged molecules (cations) will move toward the cathode, the negatively charged electrode. Electrical potential, or voltage, of the field, is directly related to the force on the charged molecules. The molecule is also affected by friction based on its shape. A larger and more asymmetric molecule will have a slower rate of movement in the electrical field due to friction. The forces that affect the charged molecule can be partially controlled through choice of voltage of the electrical field, viscosity and pH of the buffer solution in which electrophoresis takes place, and properties of the support medium for the sample. During electrophoresis, current is carried between the cathode and anode by ions in the buffer solution. The voltage applied to the electrophoresis system is chosen to minimize heat and buffer evaporation, while maximizing molecule resolution. The charged molecules move through the pores of the support medium toward an opposite charge. The movement of large molecules may be restricted by pore size of the support medium. An ideal support medium does not interact with the charged molecule chemically or electrically, but acts as a filter to retard movement on the basis of size and shape. Cellulose acetate and agarose are two of the substances that can be used as a support medium for protein electrophoresis. Movement is also due to buffer flowing across the support medium. The buffer moves in the opposite direction to the flow of negatively charged sample molecules. Buffer flow affects molecules having a small magnitude of charge more so than molecules having a greater charge. Gamma globulin proteins with pI of 7.2 actually flow from the application point toward the cathode. Although gamma globulins are negatively charged at buffer pH 8.6, the buffer flow is strong enough to carry these molecules against the electrical force. This process in which a solution moves relative to an adjacent stationary substance when placed in an electrical field is called electro-osmosis, or endosmosis. Figure 316 provides a simple illustration of electrophoresis. Following electrophoresis, the protein fractions must be fixed in the support medium to prevent their loss during the staining process. The medium can be

soaked in dilute acetic acid or trichloroacetic acid, or can be heated to denature the proteins. Proteins within an agarose medium can be precipitated out in a saline solution. This process fixes the fractions in the matrix of the support medium. The fixed medium is ready for staining. Although many charged substances that were present in the sample have undergone electrophoresis through the medium, clinical laboratory scientists/medical technologists generally examine specific categories of substances during one test. The staining procedure allows the clinical laboratory scientist the opportunity to visualize one set of substances with specificity. Proteins may be stained with ninhydrin, which will visualize amino groups; ponceau S, which will nonspecifically help to visualize proteins on a cellulose acetate medium; Coomassie brilliant blue or amido black, which are specific for proteins; or Sudan black or oil red O, which can be used to visualize lipoproteins. After staining, excess stain is removed and the background of the support medium is cleared with a solvent. The medium is then dried and ready for analysis. Once the medium has been stained and the background of the medium support has been cleared, the electrophoretic pattern can be scanned through a denO O O O Cathode Anode Power source

Figure 316. Electrophoresis.

sitometer. Densitometry works much the same way as a spectrophotometer; the stained electrophoretic pattern is scanned with a specific wavelength of light. The specific wavelength is chosen based on optimal absorbance by the stained protein. Depending on the density of the protein fraction, light is transmitted through to a photodetector that senses differences of light transmittance. Following Beers law, the amount of light absorbance is proportional to the concentration of the protein in the fraction. As the densitometer scans the electrophoretic pattern at a specific wavelength, it produces a tracing, or graph, of the relative amounts of the protein fractions present. The densitometer will also print the percentage of the total that is detected for each protein fraction by a process called integration. The process of densitometry is standardized. A gel that contains standard bands is scanned through the densitometer. The bands are standardized to transmit 10%, 20%, 40%, 60%, 80%, and 100% of the light source. As the light passes through the calibration standard, the operator adjusts electrical gain to correct transmittance to the standard percentage. As the densitometer scans the electrophoretic pattern, a tracing of the relative amounts of each protein band is produced. The densitometry report includes the electrophoretic tracing and calculation of the percentage of total protein that is detected for each protein fraction. If the concentration of total protein in the specimen is given, the absolute values of each protein fraction can be computed. Test Methodology 313 is a procedure for protein electrophoresis.

TEST METHODOLOGY 3-13. PROTEIN ELECTROPHORESIS

The Reaction Principle
The patients serum is placed into a sample trough within agarose gel, then placed in an alkaline buffer solution, and a standardized voltage is applied to allow separation of the

major protein groups by electrophoresis. The result is six or more fractions of separated proteins, including the main fractions of albumin and alpha 1, alpha2, beta1, beta2, and gamma globulins, from anode to cathode. Following electrophoresis, the agarose gel is processed in acetic acid and alcohol washes to fix the proteins. After a wash step, the protein fractions are stainined with Coumassie brilliant blue protein stain. After a second wash, fixed protein bands can be visualized and quantified with densitometry, in which light passes through the fixed medium and is absorbed by fixed stained protein bands.

The Calculations

% Protein fraction _ total protein (g/dL) _ protein fraction (g/dL) For example, if the densitometer scans and measures relative amounts of protein fractions in an electrophoretic pattern for a specimen and the absolute amount of total protein is 8.0 g/dL, then the absolute concentration of each protein fraction can be computed as shown below. Protein Fraction Relative % Absolute Concentration (g/dL) Albumin 62.5 0.625 _ 8.0 _ 5.0 Alpha1 globulin 3.75 0.0375 _ 8.0 _ 0.3 Alpha2 globulin 8.75 0.0875 _ 8.0 _ 0.7 Beta globuin 10.0 0.1 _ 8.0 _ 0.8 Gamma globulin 15.0 0.15 _ 8.0 _ 1.2 Total 100 8.0