Chng 2- in di

2.1 Nguyn tc ca phng php da trn chuyn ng ca cc phn t tch in trong dung dch khi c a vo in trng (H. 1). V cc phn t trong in trng chuyn ng vi vn tc khc nhau ph thuc vo: in tch, hnh dng, kch thc, phng php c pht trin mnh tch chng.

Phng php kh n gin v nhanh, c s dng phn tch v tch: cc phn t rt ln nh protein v axit nucleic, cc phn t tch in khc nh hn nh ng, axit amin, peptide, nucleotide, cc ion n gin.

Cc phng php pht hin c nhy cao c pht trin gh v phn tch cc kt qu tch.

Cch tin hnh:

a mt lp mu mng vo dung dch c n nh bng mt cht gi c cu to nhiu khe h (mng li). Di tc dng ca hiu in th, cc phn t khc nhau trong mu s di chuyn qua cht gi vi vn tc khc nhau. Cht gi ny l cn thit v dng in khi i qua dung dch in di s to nhit (l nhn t gy ra s khuch tn) v trn ln cc bng khi thiu mi trng to cn bng.

Cht gi c th c cu to t mt s vt liu khc nhau:

giy, acetate cellulose, polyacrylamide, agarose, tinh bt.

Trong cc gel polyacrylamide v agarose, cht gi cn c tc dng sng lc (ry) la chn theo kch thc tch. Cc phn t sau khi tch c th c pht hin di dng cc bng (vt) cc v tr khc nhau trong cht gi bng cch:

nhum s dng phng php phng x t ghi, c nh lng bng cch qut (scan) trn mt k, gel c lmg kh bo qun lu di.

Gel polyacryamide v agarose (hnh 1.2) l mi trng to n nh hay s dng nht trong cc phng th nghim nghin cu. Gel thng c to thnh dng:

thi (trong ng), bn l mng

tch protein: gel polyacrylamide l cht gi thng thng nht, tch axit nucleic: c th dng mt trong hai loi gel k trn tu thuc vo kch thc ca cc phn t cn tch.

Vic la chn cht gi v nng (gel) nh hng ln s tch da trn kch thc.

Trong phn ln cc my in di, gel c t gia hai bung cha m dng in c ni qua gel gia chng.

Mi tip xc gia m v gel c th l tip xc lng trc tip (H. 1.3 A, B, C) hoc qua mt bng giy hoc gel. (H. 1.3 D).

Gel ng ng hoc bn (H1.3 A, B) (c tip xc m trc tip) l hiu qu nht v s dng in trng

Gel c th c kch c khc nhau, tu thuc vo mc cn tch v lng mu. Cc gel ng phn tch thng c trong cc ng thy tinh vi ng knh trong 1- 5 mm v chiu di 525 cm. Cc gel ng ch phm c th n ng knh 10 cm ph hp vi lng ln nguyn liu. Ngc li, cc gel trong ng mao dn (c ng knh 50100 m v di 30- 100 mm) cho php:

c phn gii rt cao tch nhanh cn mt lng mu rt nh

Gel bn thng c gia mt cp bn knh. Bung m c to nn bng cch tch hai bn knh gia hai thanh spacer (to b dy) hai u, sau trm cc u v y gel to bung kn (H. 1.4).

Gel bn c kch thc t 2,5 cm2 (gia hai lamen ca knh hin vi) n 30. 150 cm2 v dy t <0,05 mm n > 10 mm.

Cc thng s dng in
Lc dn c bn ca in di l hiu in th ca h thng. Vn tc ca phn t t l thun vi gradient hiu in th xung quanh. C 2 phng trnh in c bn quan trng trong in di. Th nht l nh lut Ohm: V = IR I = V/R hoc

1.

nh lut Ohm lin kt: hiu in th (V) o bng vn (V), dng in (I) o bng ampe (A), in tr (R) o bng ohm ().

Phng trnh c bn th hai trong in di lin quan n power, n miu t lng nhit thot ra trong mng; P = VI hoc

P = I2R hoc
P = V2/ R

vi P l cng sut (power- o bng watt (W).

Lng nhit ny cn c bit n nh nhit o bng Joule.

Trong mch in di, in th v dng in c ngun in DC cung cp, dy ch, in cc, m v gel l nhng resistor n gin.

Ngun in dng cho in di gi mt thng s (hoc dng, hiu in th, hoc cng sut) c nh.
Tuy nhin, in tr ca mch khng c nh trong qu trnh chy. in tr m gim cng vi s tng ca nhit do nhit (Joule). in tr cng thay i khi vng ion m ngt qung chuyn ng qua gel , trong trng hp ca SDS- PAGE ngt qung, in tr tng khi chy. Ph thuc vo m v thng s no c gi nguyn, nhit Joule ca gel c th tng hoc gim trong thi gian chy (B. 1... )

- m v pH

Protein l cc cht lng tnh, do vy c th tch in m hoc dng, v chng c cha c hai loi gc acid v base. Axit nucleic khng phi lng tnh v tch in m a s m in di (do bn cht axit mnh ca cc nhm phosphate ca b khung). a s in tch protein c c l t s ion ho (ph thuc pH) ca cc nhm carboxyl v amino ca mch bn: - COOH COO- + H+ - NH2 + H+ NH3+

His- mt a.a kim yu cng c cha in tch gn pH trung tnh. V nhng nhm ny c th chun trn vng pH in di bnh thng, in tch thc (net) ca 1 protein c xc nh bng pH ca mi trng xung quanh, s v loi a.a mang nhm carboxyl v amine Mi protein u c pI ring (nh Hb ca ngi l 7,07, lactoglobin l 5, 34, casein b l 4,7). Trong mt dung dch c pH ln hn pI, protein c in tch m (thc) r rt v s di ng v cc dng (anode) trong in trng. Cn nu pH nh hn pI th s ngc li. tch protein bng in di da trn s di ng ca cc phn t khc nhau, pH dung dch cn c gi n nh duy tr

- Tc ng ca nhit ln qu trnh tch

S iu khin nhit l then cht trong mi bc ca in di. V d, s polymer ho ca acrylamide l 1 phn ng to nhit v nhit to ra c th gy ra s i lu dn n khng ng u trong kch thc mng li trong gel (c bit i vi gel nng cao). Nhit cao c th gy ra nhiu vn :

nhit qu cao c th khin gel agarose b tan (chy) ra, bn knh b v, hoc gy h hi n thit b in di cc protein c th b bin tnh v bt hot. c th khc phc cc hin tng trn bng cch s dng cc thit b lm lnh

- Cht gi Gel agarose v polyacrylamide l nhng cu trc c lin kt ngang ging nh bt bin.

Mc d chng cha n 99% nc, kch thc mng li ca chng ging vi kch thc ca protein v axit nucleic.
V cc phn t b hiu in th buc phi chuyn ng qua gel, cc phn t ln s i chm hn cc phn t nh. i vi 1 gel nht nh, nhng phn t nh hn kch thc do gel quy nh s khng b chm li v chuyn ng gn ging nh trong dung dch t do. Ngc li, nhng phn t ln hn gii hn quy nh ca gel s khng chui vo bn trong gel c.

C th to ra gel vi kch thc mng li ( lin kt) to thnh ry phn t bng cch chn nng thch hp ca gel. Kch thc mng li ca gel ph thuc vo phn trm cht rn trong gel, v i vi polyacrylamide l lng lin kt ngang v tng lng polyacrylamide s dng.

- Gel agarose:
Agarose l dn xut polysaccharide t agar (thch) c tinh khit cao.

C rt nhiu loi agarose, khc nhau v:

in mi, nhit nng chy, cng trong.

s dng cho mc ch sinh hc, quan trng l EEO thp v trong nng cn dng.

Agarose thng c cung cp di dng bt kh. Khi cho vo m si, n s tan v gi c trng thi lng cho n khi to gel nhit c h xung khong 40oC. Khi ng, gel s n nh nhit thp hn. (C nhng loi agarose c bit c nhit nng chy v to gel thp hn ng k, cho php thu li mu t gel sau khi tch v x l tip tc bng enzyme nu cn). Kch thc mng li v c tnh sng lc ph thuc vo nng agarose trong gel. Nng cng cao, mng li cng nh. Thng nng thch hp cho s dng l t 0,4% n 4% (w/v).

- c im ca in di trn gel agarose:

1. 2.

ng dng tch v phn tch AND La chn h thng m, nng gel, iu kin chy (dng in, thi gian, nhit ...) thch hp S dng EtBr v tia UV pht hin cc bng ADN trn gel.

3.

- in di trn gel polyacrylamid

in di trn gel polyacrylamide c SDS (SDS- PAGE): tch protein da trn KLPT (MW). Khi tch bng SDS PAGE, s di ng c xc nh khng phi do in tch ca chui polypeptide, m do khi lng phn t (MW). SDS (sodium dodecylsulfate) l mt cht ty anion gy bin tnh protein bng cch bao quanh b khung polypeptide khin phn t dui thng. Mi phn t SDS s cung cp hai in tch m, cc phn t protein s c tch in t l vi khi lng phn t ca chng

SDS-PAGE

Trong in di bin tnh trn gel polyacrylamide (in di c cht kh), di ng ca cc phn t c xc nh khng phi bng in tch ca protein, m bng khi lng phn t ca chng. SDS (sodium dodecylsulfate) l 1 cht kh anionic gy bin tnh protein bng cch bao quanh khung polypeptide v tch in m cho n t l vi chiu di ca mch. Khi c x l bng SDS v cht kh, cc protein u c dng hnh cu tch in m vi cng s in tch trn 1 n v chiu di.

phn gii ca SDS-PAGE l rt ln: cc phc hp c th c tch thnh hng trm vt (bng band) trn gel.

V tr ca bng dc theo ging cho bit gi tr gn ng v kch thc v lng protein trong mu (cn c vo bt mu sau khi nhum ), da vo c th nh gi v:

sch, mc biu hin, in chuyn min dch, chun b phn tch trnh t, to cc khng th nh SDS-PAGE.

C hai kiu h thng m trong in di: lin tc v ngt qung (discontinuous).

H thng lin tc ch c 1 gel tch duy nht v s dng cng 1 loi m cho gel v chy in di.

Trong in di ngt qung (Ornstein v Davis, 1964), 1 lp gel c mng li ln (long hn) gi gel c c ln trn lp gel tch. Mi lp c 1 loi m ring, v khc vi m chy. Tuy h thng u (lin tc) d chun b hn, t b ta mu v b ngng tp khi chy hn, nhng phng php sau li cho phn gii cao hn nhiu.

Trong in di ngt qung, di ng ca 1 protein (s o tc ca 1 phn t tch in trong in trng) l tr s trung bnh gia:

di ng ca ion m trong gel c (leading-ion dn, i trc) v di ng ca ion trong bung in di trn (trailing-ion- c ko theo sau).

Khi in di bt u, cc ion v protein bt u di ng vo gel c. Protein c c c li trong 1 vng rt mng, gia ion dn v ion c ko theo, v tip tc di ng trong gel c cho n khi n c gel tch. Ngc li, ch c 1 c c ti thiu vi h thng lin tc v cc protein c tch thnh 1 vng cng rng gn bng dy ca mu lc ban u trong ging,

Figure 4.7 Electrophoresis- The loading of samples

Figure 4.8 Electrophoresis The protein bands after treating the gel with Coomassie blue stain.

Figure 4.9 Estimating the molecular weight of a protein.

The electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight, Mr.

- in di phn vng ng in protein

Gii thiu chung -in di hai chiu (2D-PAGE) Gii thiu chung ng dng ca 2D-PAGE

Isoelectric focusing is a procedure used to determine the isoelectric point (pI) of a protein (Figure ). A pH gradient is established by allowing a mixture of low molecular weight organic acid and bases (ampholytes) to distribute themselves in an electric field generated across the gel. When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pI. Proteins with different isoelectric points are thus distributed differently throughout the gel (Tab ).

Two dimensional electrophoresis

The combination of these two different electrophoretic methods in twodimentional gels permits the resolution of complex mixture of proteins (Fig ) and with more sensitivity. Two-dimentional electrophoresis separate proteins of identical molecular weight that difer in pI, or vice versa

Two dimensional electrophoresis has several aims: Proteins separated by electrophoresis are then identified by affinity- or immunoelectrophoresis according to Laurell (1966) Part of the fractions of a complex protein mixture are separated by one method and then further purified by a second method based on other separation principles in order to prevent the interference of irrelevant proteins. Mixture of proteins are separated so that the physico-chemical parameters, such as pI and the molecular weight, can be read on the 2D electropherogram as on a coordinate system. Complex mixture of proteins such as cell lysare or tissue extract should be completely fractionated into indivudual proteins so as to obtain and overall picture of the protein composition and to enable location of indivudual proteins.

For these methods, the first-dimentional runs are carried out in gel rods or strips and loaded onto the seconddimentional gels. A flat-bed gel can also be cut into strips after the first separation and transferred onto the second gel. In most cases the first-dimentional gel must be equilibrated in the buffer for the second-dimentional run to recharge the protein, for example to unfold the secondary and tertiary structure and to form a micelle with SDS. High resolution 2D electrophoresis (O'Farrell and Klose (1975)) (now a "classic" method), the first dimention is isoelectric focusing in presence of 8 or 9 M urea -close to the saturation limit- and a non-ionic detergent such as Triton X-100 or Nonidet NP-40. The second dimention is and SDS electrophoresis. The separation parameter of the first-dimentional run- the pI- is indipendent of the molecular weight which is the separation parameter of the second dimentional run

The result of the separate is a pattern of spots. According to to the Cartesian coordinate system, the following standard is accepted: from left to right- increasing pI, from bottom to top- increasing molecular weight. These two dimentional protein maps afford the highest resolution of all the methods at the moment. Autoradiography of labelled proteins and silver stainning are used most often. The use of immobilized pH gradient (IPG-Dalt) for the firstdimentional run has increased the reproducibicity of the pattern and allows to detect the basic proteins. Even extremely wide immobilized pH gradients, e.g. pH 2.5 to 11, can be used, in order to separate nearly all possible cellular products in a single two-dimentional map. Evaluation of 2D patterns is performed with a computer. The pherogram are therefore converted into digital signal with densitometer, high resolution desk top scanners or video cameras. With the appropriate evaluation software the data are processed for qualitative and quantitative analysis.

Figure 4. 10 Two-dimentional electrophoresis

Figure 4. 10 (cont) Two-dimentional electrophoresis

Proteome analysis
Antibody microarray (Protein Chip)

Proteome analysis 2D Electrophoresis

Principle

Silver stained 2D gels

Proteome analysis

Protein annotation by mass spectroscopy

K thut chuyn thm protein (Western blot) v kt ta min dch

1. Nu khi nim chung v nguyn tc ca cc phng php chuyn thm (blot): Southern (cho AND), Northern (cho ARN), v Western (cho protein). 2. Cc loi mng v thit b cn thit 3. Chuyn protein t gel SDS-PAGE sang mng 4. H thng chuyn dng bn cha 5. H thng chuyn bn sy 6. Cc phng php pht hin khng th 7. Pht hin protein tng s trn mng chuyn: gii thiu cc phng php pht hin cc cht trn mng thm: lai bng mu d (vi axit nucleic), khng th c hiu (vi protein), s dng