CellMosaic has designed this AP-oligo conjugate with linkers and conjugation chemistry that retain the activity of AP. The same configuration and chemistry has been tested for AP-oligos with different oligo sequences from a few diagnostic companies since 2011.
The kit includes purified maleimide activated AP that 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 was chosen to enhance aqueous buffer solubility. The Q-column purification step typically provides the resulting AP-oligo at greater than 95% purity.
This kit provides materials to label one (CM53403x1) to three oligo samples (CM53403x3). Scale of the reaction: 2.5 nmol oligo.
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Key Features of this AP-Oligo Conjugation Kit:
- Offers conjugation and purification steps that primarily provide single-labeled products with minimum background signal from the free (unreacted) AP
- High quality activated alkaline phosphatase (>95% pure)
- Fast preparation: less than 4 h hands-on time
- No AP activity loss during the conjugation and purification
- Quality of the conjugate: >90% of labeled conjugates with majority single-labeled based on size-exclusion chromatography (SEC)
- Stable and long flexible PEG-based linkage to minimize the interaction between AP and oligo
- 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 SEC and Anion exchange HPLC analysis.
Typical Kit Performance Data (LC Analysis, CellMosaic)
Oligo information: HPLC-purified 23mer oligo with 5’ NH2-C6 modification
Kit lot number: 5527.s12.070819
SEC HPLC profiles of AP (blue trace), oligo (green trace), and purified product (E1) (red Trace) detected at 220 nm. SEC separates the conjugates by apparent molecular weight (MW) or size in aqueous solution. The larger the MW of the conjugate, the earlier it elutes.
Figure 2: Anion exchange chromatographic HPLC profiles of the AP (blue trace), oligo (green trace), and AP oligo conjugation reaction mixture (red Trace) detected at 220 nm.
Table 1: Summary results
|Sample||Volume µL||% free AP||% free oligo||% of AP/oligo (1:1)|
|E1 (first elution)||400||Not detected||Not detected||93%|
|Recovery: 52% of the conjugates|