AP Oligo Conjugation Kit (Single Label with Amine Oligo)

Product Description

CellMosaic® has designed this AP-oligo conjugation kit with linkers and conjugation chemistry that retain the activity of AP. The same configuration and chemistry has been tested for AP-oligos prepared with different oligo sequences from several diferent diagnostic companies since 2011.

The kit includes purified maleimide activated alkaline phosphatase (AP) prepared from highly active and EIA grade AP from Calf intestine (>3000 U/mg). The user supplies the amine-modified oligos, which are readily available from many commercial oligo suppliers. Using the kit components, the user converts the amine-oligo to a thiol-oligo, followed by reaction of the thiol-oligo with activated AP to generate the AP-oligo conjugates. A hydrophilic PEG linker enhances aqueous buffer solubility and minimizes any interaction between AP and the oligo. The Q-column purification step typically provides the resulting AP-oligo at greater than 95% purity.

This kit provides materials to perform Alkaline Phosphatase (AP) labeling of one or three oligo samples. Scale of the reaction: 2.5 nmol of oligo.

If you purchased this kit previously, the current one is an upgraded and improved kit with slightly different components and workflow as the first-generation kit. This kit has multiple configurations for various lengths of oligos and can be used to conjugate 2.5 nano-mole (nmol) of oligo. Table 1 provides the catalog numbers for various kit configurations.

Table 1: Configurations of the PerKit™ AP-Oligo conjugation kit (CM53403)

Requirement for amine-oligo:

• Amount: 2.5 nmol 
• Preferably, the amine-oligo should be HPLC purified and lyophilized
• Additional components of acid or other molecules containing primary or secondary amine
  groups and thiol should NOT be present

Key Features of this AP-Oligo Conjugation Kit:

• High quality activated alkaline phosphatase (>95% pure). 
• Fast preparation: less than 5 hours of hands-on time. 
• Minimum or no AP activity loss during the conjugation and purification.
• A single purification affords 85-90% single-labeled HRP-oligo conjugates if the quality of the oligo is at par.
• Stable linkage with optimal spacer to ensure no interference between oligo binding and AP activation.
• All reagents included, from preparation to purification
• Options to choose tailored services at CellMosaic® after conjugation

After conjugation: you can choose to send your sample to CellMosaic® for size-exclusion chromatography (SEC) and anion exchange (AEX) HPLC analysis. See sample data below.

SEC HPLC separates the conjugates by apparent molecular weight (MW) or size in aqueous solution. The larger the MW of the conjugate, the earlier it elutes.

AEX HPLC separates the components according to the negative charges of the molecule. 

             You can choose to send your conjugates to CellMosaic for complete removal of trace oligo impurities and unreacted AP.

 Labeling Chemistry

Typical Kit Performance Data (LC Analysis, CellMosaic®)

Oligo information: HPLC-purified 23mer oligo with 5’ NH2-C6 modification

Kit lot number: 5527.s12.070819

Figure 1: SEC HPLC profiles of maleimide activated AP (blue trace), oligo (green trace), and purified product (E1) (red trace) detected at 220 nm. 

Figure 2: AEX HPLC profiles of the maleimide activated AP (blue trace), oligo (green trace), and AP oligo conjugation reaction mixture (red trace) detected at 220 nm.

Table 1: Summary results