Introduction

As a researcher you are often required to distinguish the special form of a protein (mutated or post-translationally modified) from its normal counterpart.  For this purpose, antibodies would be raised against a short sequence that contains a specific site of alteration or modification.  Scientists at Li International are well aware of the technical difficulties and high cost that you might face in this process.  We are thus pleased to introduce the AccuPoint program with the twin goals of lowering your investment and maximizing your chance of success.

Types of Modification Detected by Antibodies

AccuPoint is designed to provide you good antibodies (both monoclonal and polyclonal) against proteins with methylated and acetylated lysines, phosphorylated serines, threonines and tyrosines, as well as point mutations (see Table 1).  Thanks to our expertise in the synthesis of special peptides, this service also enjoys a high success rate with other, less common types of modifications.

Procedure

A typical project starts with the synthesis of 2 peptides: an antigen peptide XXXXXXMXXXXXX where M stands for the modified (or mutated) amino acid; and a control peptide XXXXXXAXXXXXX where A stands for the unmodified residue.

The antigen peptide is used to immunize mice (in a monoclonal project) or rabbits (in a polyclonal project).  The antibody generation processes resemble those in the AccuMono or AccuPoly program.  However, as described below, additional screening steps are required to eliminate non-specific antibodies (i.e., those reacting with both the modified and the unmodified protein).

Further Screening of Polyclonal Antibodies

We would first immunize rabbits with the antigen peptide and harvest polyclonals.  The antibodies then go through two affinity columns: the first one contains the antigen peptide.  Therefore, all antibodies binding to the modified protein are collected on this column.  The captured antibodies are then passed through a column with the control peptide.  In this step, non-specific antibodies (i.e., those binding both the modified and unmodified protein) are removed.

Further Screening of Monoclonal Antibodies

Using an ELISA assay, we would first measure the affinity of each antibody (monoclone) to the antigen peptide. Only clones with good "signals" (i.e., ELISA titer > 100,000) are selected for further screening.  In the second round of ELISA assays, candidate clones from the first round of screening are tested against the control peptide, and those with relatively high affinity (to the control peptide, i.e., "noises") are discarded.  A good antibody would bind the antigen peptide much more strongly (> 8 times) than the control peptide.

Pricing

Please contact us for an evaluation.