Biomedical Engineering Reference
In-Depth Information
A
18.25 g acrylamide, 0.487 g methylene-bisacrylamide in 50 ml
Solutions/Reagents
ddH
2
O
B . 2M KOH, 0.75 M acetic acid, pH 4.3
C . 2M KOH, 0.125 M acetic acid, pH 6.8
D . 5M
β
-alanine, 0.14 M acetic acid, pH 4.5
E
10% TEMED (v/v) in ddH
2
O
F
10% ammonium persulfate (w/v) in ddH
2
O
The pipetting scheme is given in Table 2.9. Hints for preparing
the gel are described in Protocol 2.1.8. Sample as well as electrode
buffer is Soln. D.
References
Reisfeld TA, Lewis UJ, Williams DE (1962) Nature 195:281
2.1.10 Affinity Electrophoresis
The principle of affinity electrophoresis is a nondenaturating (“na-
tive”) electrophoresis using a matrix containing a ligand or receptor
interacting with some of the separated macromolecules resulting
in changed mobility of the complex (ligand-ligate interaction). It is
possible to analyze whether a protein (e.g., glycoprotein) interacts
generally with another macromolecule (e.g., lectin; cf. Table 2.19).
By variation of the ligand or ligate concentration it is possible to
estimate the dissociation constant of the complex.
Usually a non-covalent immobilized reactant is incorporated
into the gel and the electrophoretic conditions are chosen in that
way that a significant motion of this reactant does not occur.
Especially in the case of low molecular mass ligands these
ligands are covalently attached to the gel matrix, e.g., by co-
polymerization of allylesters of mono- or oligosaccharides.
A special case of affinity electrophoresis is immunoelectropho-
resis (cf. Chap. 4.10), which analyzes antibody-antigen interactions.
Horizontal Affinity Electrophoresis in Agarose Gels
A
1.5% agarose standard EEO (w/v) dissolved in ddH
2
Ofor2-
Solutions/Reagents
3 h at about 80
◦
C
=
B
barbital-acetate buffer I
0.05, pH 8.6 (cf. Protocol 7.4.1)
PBS
The ligand is dissolved in Soln. B with a concentration of 0.2 -
1 mg
/
10-cm plate 5 ml are needed). Heat this solution
to 60
◦
C and mix with an equal volume of agarose solution A, also
heated to 60
◦
C. Pour the mixture onto a horizontally straightened
glass plate. Introduce the comb and allow the gel to solidify at room
temperature.
Mix the samples with Soln. B in a 1:1 ratio; supplement some
bromophenol blue.
ml (for a 10
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