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Use of ZipTip Pipette Tips for Microscale Affinity Mass Spectrometry

Overview

Affinity chromatography forms the basis of the separation and purification of a number of biological molecules. For example, antibodies may be purified based on their affinity for protein A, or a protein may be purified on the basis of its affinity for a monospecific antibody raised against it. Typically, these purifications take place in a chromatographic column with a relatively large volume. This note discusses the use of the C18 ZipTip pipette tips for conducting affinity-based separations on a microliter scale.

Key Features

Affinity Separation Protocol

Examples of ZipTip Pipette Tip Affinity Separations

Experimental Conditions

Accelerating Voltage: 25 kV Grid Voltage: 93%

Mode: Linear Guide Wire Voltage: 0.1 to 0.3%

Matrices: Sinapinic acid (SA) at 10 mg/mL in 50% MeCN/0.2% TFA, α-cyano-4-hydroxycinnamic acid at 10 mg/mL in 50% MeCN/0.2% TFA.

Affinity Separation Protocol

Wash ZipTip pipette tip (MeCN, 50% MeCN, 0.1% TFA)

Incubate ZipTip pipette tip with Affinity Agent, e.g. Ab

Wash with incubation buffer

Incubate ZipTip pipette tip with blocking agent, e.g. BSA

Wash with incubation buffer

Incubate with sample, e.g., antigen Wash with incubation buffer, then water

Elute sample with acid, e.g., 1% TFA Elute with 50% MeCN

We followed the affinity separation protocol above. After conditioning the ZipTip pipette tip (Figure 1), a goat anti-human transferrin was incubated with it by passing 20 μL of anti-serum up and down the tip ten times. After 3 washes with 10 μL aliquots of phosphate buffered saline at pH 7.4 (PBS), 20 μL of a 20 mg/mL solution of bovine serum al
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