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2.聚合物在蛋白质分离中的应用 PowerPoint PPT Presentation


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2.聚合物在蛋白质分离中的应用. 蛋白质是由氨基酸通过酰胺键连接而成的高分子,两性,存在等 电点,因其空间结构的复杂性,有亲水或疏水之分。. 蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷. 若缓冲溶液的 pH 值低于蛋白质等电点,蛋白质则吸附 H + 而带正电荷,由于 静电相互作用,蛋白质与管壁发生 吸附。. ( a )碱性蛋白质( pI = 8 )所带电荷量随 pH 的变化曲线; ( b )在 pH = 4 时,碱性蛋白质( pI = 8 )与毛细管壁发生吸附. 1 . 极端 pH 值法.

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2.聚合物在蛋白质分离中的应用

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  • pHH


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apI8pH

bpH4pI8


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. pH

  • pHpIpH410pH11.0

  • pH1.5

  • pHpKa

McManigill D. Anal Chem,198658166170.


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2.

  • N-N,N, N,N--1,3 -TMBD

  • pH

.. 1996, 7275.

Verzola B, Gelfi C, Righetti P G. J Chromatogr A20008688599.

Corradini D, Cannarsa G, Fabbri E, et al. J Chromatogr A1995709127134.

Corradini D, Cannarsa G. Electrophoresis199516630635.


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.

(1)

PVA42902

50ug/mL1C23450m48.5cm40cm3 kV, 5 s40mM309 V/cm


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(2)

DNA

N,N-

Madabhushi R S. Electrophoresis199819224230.

Verzola B, Gelfi C, Righetti P G. J Chromatogr A2000874293303.


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(1) Cat-HEC

Formation of the cat-HEC

Schematic diagram of adsorption of cat-HEC onto silica surface.

Preparation and properties of Cat-HEC


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Electroosmotic flow as a function of pH. Comparison between

a bare fused-silica capillary, HEC and cat-HEC coated capillary

Electropherograms of a mixture of standard proteins.

Separations were carried out using cat-HEC-2 coated capillary

1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.

Electropherograms of a mixture of standard proteins at pH 4.6.


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Electropherograms of a mixture of standard proteins in different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.

Migrationtime reproducibility (n = 3) and peak efficiency of proteins separated in polymer-coated capillaries at pH 4.6

Electrophoresis,2008, 29, 1460-1466.


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() HEC-g-P4VP

Electroosmotic flow as a function of pH. Comparison between

a bare fused-silica capillary, HEC and HEC-g-P4VP coated capillary

Formation of the HEC-g-P4VP

Electropherograms of a mixture of standard proteins. Separations were carried out using cat-HEC-g-P4VP coated capillary. 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.


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O

O

O

B

r

O

B

r

+

O

O

N

N

N

PEO-b-P4VP

Formation of the PEO-b-P4VP

1H NMR and GPC data of PEO-b-P4VP copolymers

a. Mn of PEO-b-P4VP estimated by 1H NMR b. Mn of PEO-b-P4VP determined by GPC.


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(A) TypicalTEM images of (a) PEO113-b-P4VP45, (b) PEO113-b-P4VP90,

(c) PEO113-b-P4VP113, and (d) PEO113-b-P4VP294;

(B) Schematic illustration of copolymers of different morphologies capillary coating procedure

(a) PEO113-b-P4VP45, (b) PEO113-b-P4VP90, (c) PEO113-b-P4VP113, and (d) PEO113-b-P4VP294

Effect of molecular weight of P4VP block on the separation of basic proteins at pH 4.6.

1, lysozyme; 2, cytochrome c; 3, ribonuclease.


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Analysis of saliva samples by PEO113-b-P4VP294 coated capillary.

Saliva samples were diluted to 2-fold using deionized water. Samples (A)

without and (B) with spiking of 0.2mg / mL lysozyme were injected.

Separation conditions: 40mM phosphate-citrate buffer, pH 5.1; 500V/cm;

40cm capillary (30 cm to the detector); temperature 25.

Effect of buffer pH on the separation of basic proteins.

separations were taken in PEO113-b-P4VP294 coated capillary


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Electrophoresis 2008,

29, 2812-2819

Electropherograms of plasma sample in bare capillary (A) and a PEO113-b-P4VP294 coated capillary (B).

Separation buffer: 19mM NaOH-Na2B4O7 at pH 9.7. HSA, human serum albumin; 1-AT, 1-antitrypsin; 2-M, 2-macroglobulin; -lp, -lipoprotein; Tf, transferrin; IgA, immunoglobulin A; IgG, immunoglobulin G.


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3.

DNA

(1) HEC-g-PDMA

Electrophoresis,

2008, 29, 43514354.

Separation of 174/HaeIII digest by CE with

HEC-g-PDMA at (a) 1.0% w/v; (b) 1.5% w/v; (c) 2.0% w/v

Effect of pH values on separations of basic proteins using

HEC-g-PDMA coated capillary.1, lysozyme; 2, cytochrome c; 3, ribonuclease A.


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(2) quasi-interpenetrating network of LPA andPDMA

Properties of quasi-IPN containing LPA with different molecular masses

EOF as a function of pH.

Electropherograms of a mixture of standard proteins.


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Separation of basic proteins using quasi-IPN-3 coated capillary

at different pH values. 1=cytochrome c; 2=Lysozyme; 3= Ribonuclease A.

J. Sep. Sci., 2009, 32, 671-680.

Separation of 174/HaeIII digest by CE with quasi-IPN-3 at

(a) 2% w/v; (b) 3% w/v; (c) 4% w/v; (d) 4% w/v. Conditions: Separation electric

field strength of (a), (b), and (c) is 6 kV, separation electric field strength of (d) is 8 kV.


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  • pHpH


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Thank you !


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