UNIMO Technology UDR-204 Manual de instrucciones descargar pdf (Pagina 162)

Fluid Operation

Lysozyme on Mica—A Model Procedure for Protein Binding

142 MultiMode SPM Instruction Manual Rev. B

8.6 Lysozyme on Mica—A Model Procedure for Protein

Binding

8.6.1 Protein Binding Theory

All proteins contain free amino groups that become positively charged at sufﬁciently low pH. If

sufﬁcient free amino groups are located on the outside surface of the protein, then the protein will

bind to a negatively charged mica surface. Proteins will become positively charged at pH below

their isoelectric point and are then able to bind to mica. The protein lysozyme, for example,

becomes sufﬁciently positively charged to bind to mica at pH 6. This is shown schematically below

in Figure 8.6a.

Figure 8.6a Proteins will typically bind to negatively charged mica when the pH is reduced below the protein’s

isoelectric point, pI

8.6.2 Protein Binding Procedure

The following section gives a detailed procedure for preparing and imaging the protein lysozyme

by TappingMode in ﬂuid. The procedure was kindly provided by Monika Fritz at the University of

California, Santa Barbara and is described in the following paper:

• Radmacher, M., M. Fritz, H.G. Hansma, P.K. Hansma (1994). “Direct Observation of

Enzyme Activity with Atomic Force Microscopy.” Science 265, 1577.

1. Obtain the required materials:

• Deionized water

• Mica substrates

• Lysozyme protein L-6876 from Sigma Chemical

• Phosphate buffer solution, 10 mM KH

, 150 mM KCl, pH 6 (buffer may be

adjusted for other proteins)

Mica

Buffer

Proteins

pH < pI

1 2 ... 157 158 159 160 161 162 163 164 165 166 167 ... 357 358

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UNIMO Technology UDR-204 Manual de instrucciones Pagina 162