Cellulose Acetate Membrane Electrophoresis

xiaoli

Serum protein

electrophoresis

(CAE)

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Its principle is that the charged particles of a sample migrate in an applied electrical field.

What is Electrophoresis ?

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It is applied for the separation and characterization of proteins, nucleic acids and subcellular-sized particles like viruses and small organelles. Electrophoresis is an analytical method frequently used in molecular biology and medicine.

What is function of Electrophoresis ?

Analysis at the level of macromolecules often involves isolation and purification of a particular one from among hundreds or thousands, assaying specifically for this molecule, determining size (molecular weight), shape, and chemical structure.

Electrophoresis is used to identify and study charged molecules and macromolecules.

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The electrophoretic mobility v of a molecule is defined as the migration per unit field strength: v = v/x = q / 6πη r

electrophoretic mobility (v)

molecule of charge q; electric field of strength x; medium of viscosityη; a spherical molecule of radius r ; a spherical molecule of radius r

velocity v

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The migration of length per time:

v : cm / min

Migration velocity depends on

Strength of electric field (Heat)

net charge on molecule (pH)

size and shape of molecules (choice of support)

Properties of supporting medium (viscosity, electroendosmosis)

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Electrophoresis of supporting medium

Sodium dodesyl sulphate-plyacrylamide gel electrophoresis (SDS-PAGE)Agarose gel electrophoresisCellulose acetate membrane electrophoresisFilter paperNative gels

Barbitone buffer (pH 8.60, 0.06mol/L)

Albuminα1-globuminα2-globuminβ-globumin γ-globumin

pI=4.9pI=5.28pI=5.82pI=7.3pI=6.8

Effect of pH and buffer on protein charge

Proteins are amphoteric compounds and are therefore either positively or negatively charged

H- C - COO-

NH3+

R

H- C - COOHNH3+

RH- C - COO-

NH2

RH+ H+

PI=pH pI<pH

Isoelectric Point - pH where there is no net charge in molecule.

PI>pH

1. Horizontal electrophoresis apparatus2. Cellulose acetate strips3. Serum4. serum applicator5. Power pack6. Barbitone buffer (pH 8.60, 0.06mol/L) 7. Whatman 3 MM paper 8. Citrate buffer (pH 6.8, 0.1 mol/L)9. Amino black 10B stain (0.1%) 10. Washing solution (Methanol: acetic acid: water = 9:1:10)

SPECIMENS & MATERIALS

PROCEDURE1. Preparation of Apparatus: Add the buffer into the chambers. Use filter paper

as salt bridge whose one end immerse in the buffer and another put onto the plastic bracket after the buffer in the different (anode’s and cathode’s) chambers have the same height.

2. Prepare the strip of cellulose acetate

varnish

frosting

Remove the strip of cellulose acetate only with forceps! On the frosting of the strip, lightly mark a beeline at 1.5cm of one end with a pencil. Then moisten a strip by placing it on the surface of the buffer in a flat dish and allow the buffer to soak from below. Immerse the strip completely by gently rocking the dish (about 10 -20 minutes).

b. Immersing the Strips:

3. Sample Applying: Remove the strip and put between two pieces of filter paper carefully, obsorbe excess buffer solution. Fit the applicator to the block and gently depress, hold down for 3 seconds to completely fill the slots, and release the applicator slowly.

4. Balancing and Electrophoresis:

Barbitone buffer (pH 8.60, 0.06mol/L)

α1-globumin pI=5.28α2-globumin pI=5.82β-globumin pI=6.8γ-globumin pI=7.3

Albumin pI=4.9

5. Staining:

When electrophoresis is completed, turn off the power and disconnect the cords BEFORE removing the chamber cover.

Remove the strips, and stain with for 5-10 min in amino black 10 B.

6. Washing and Observing:

Excessive dye from the strip is released by washing repeatedly.

Rinse in washing solution for three separate rinses of at least 2 min each with mild agitation.

Strips may be stored in 5% acetic acid in the refrigerator if necessary. Strips may be preserved for a report by blotting, air drying and taping to a report form with transparent tape.

Washing solution (Methanol: acetic acid: water = 9:1:10)

1=albumin ；　　 2 ， 3 ， 4 ， 5 is α1- ， α2- ， β- andγ-globumin ；　　 6 is origine

A 1 2

A 1 2

(a)

After scanning A 62 ％～ 71％

1 3 ％～ 4 ％

2 6 ％～ 10 ％

7 ％～ 11 ％

9 ％～ 18 ％

(b)

Clinic significance

1. Normal value:

Normal

hepatocirrhosis

hepatocirrhosis

nephrotic syndrome

Normal medullary tumour

medullary tumour

disease albumin α1globulin α2 β γ

Kidney disease ↓↓ ↑ ↑↑ ↑ ↓

Diffuse liver damage ↓↓ ↑ ↓ ↓ ↑

hepatocirrhosis ↓↓ ↓ ↓ β-γbridge

Idiopathic liver cancer ↓↓ AFP ↑

medullary tumour ↑ ↑↑

Chronically inflammation

↓ ↑ ↑ ↑

gestation ↓ ↑ ↓

No- γglobulindisease ↓↓

Double albumin disease

Double apex

Acidic and

Basic amino acid