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Amersham AlkPhos Direct™ Labeling and Detection Systems- Fast and convenient system for all high-sensitivity applications using DNA, RNA or oligonucleotide probes.
- Hybridization stringency can be controlled by elevating temperatures up to 75°C.
- Detection of 60 fg target DNA (single-copy gene) in a genomic Southern blot.
- ECF™ detection for quantitation experiments.
- Saves 2 to 3 h over conventional indirect methods.
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Amersham AlkPhos Direct™ Labeling and Detection Systems
Technical Information
Probe labeling
Hybridization
Detection
AlkPhos Direct™ Labeling and Detection Systems are based on the rapid, direct labeling of DNA or RNA probes with thermostable alkaline phosphatase. CDP-Star™ or ECF™ detection can be used.
DNA or RNA probes are labeled during a 30 min reaction and resulting probes can be used without purification. Hybridization stringency can be controlled by temperature as well as salt concentration. Detection is rapid, taking only 30 min from completion of hybridization to application of detection reagent.
Each system includes the following reagents, sufficient for 25 or 50 labeling reactions and detection of 2500 cm2 or 5000 cm2 membrane (depending on product ordered): labeling reagent, crosslinker, control DNA, reaction buffer, hybridization buffer, blocking agent, CDP-Star™ or ECF™ detection reagents.
Comparison of AlkPhos direct and hapten based system
AlkPhos Direct™
A competitor’s hapten-based system
Southern blot
Cosmid DNA digested with Not I and Eco RI, probed with a 1.1 kb probe labeled with AlkPhos Direct™ and a competitor’s hapten-based system.
Increased stringency with temperature
Customer data, maize genome with maize probe labeled with AlkPhos Direct™.
Results show blots hybridized and washed at:
50°C
55°C
60°C
65°C
What is AlkPhos Direct?
DNA or RNA probes are labeled directly with a high turnover, thermostable alkaline phosphatase enzyme (patent pending) in a 30 minute reaction. Resulting alkaline phosphatase - labeled probes can be used in hybridizations without further purification. Detection is rapid, taking only 30 minutes from completion of hybridization to application of the detection reagent. Reprobing of Southern and Northern blots can be easily achieved.
AlkPhos Direct labeling and detection systems are available with the detection reagent CDP-Star™ for high sensitivity chemiluminescent applications, or with ECF™ detection substrate for fluorescence scanning instrumentation
Rapid protocol saves time
Time savings of 2-3 hours over conventional hapten-based methods are achievable as a result of the rapid and simple labeling reaction, and the absence of probe purification and lengthy antibody development.
High sensitivity
Each batch is quality controlled to ensure detection with CDP-Star of 0.06pg of target DNA in a human genomic Southern blot.
Stable DNA or RNA probes
Both DNA and RNA probes can be labeled with this system. Probes may be labeled in bulk and are stable for several months at -20°C in 50% glycerol.
Familiar stringency control
Due to the thermostable nature of the enzyme, hybridization stringency can be controlled by elevating temperatures up to 75°C.
Quantitative
ECF substrate allows accurate quantification over 5 orders of magnitude when used with fluorescence scanning instrumentation.
How does AlkPhos Direct work?
Probe labeling
Denatured or single stranded DNA or RNA probe is mixed with the labeling buffer and alkaline phosphatase enzyme. Formaldehyde is then added to covalently cross-link the enzyme to the probe. There is no need to purify the probe before hybridization. Total labeling time: 30 min
Hybridization
The probe is hybridized to the blot using the specially formulated AlkPhos Direct™ hybridization buffer, which stabilizes the activity of the enzyme. After a 15 min pre-hybridization, hybridization is performed overnight at the required temperature. For higher target applications, a 2-4 h hybridization may be sufficient.The recommended membrane for hybridization is Hybond™-N+ (nylon).
Hybridization time:15 min pre-hybridization 2-16 h hybridization Stringency washes:30 min
Detection
i) Chemiluminescence with CDP-Star™Hybridized blots are detected with CDP-Star reagent. Following a 2 min incubation, blots are exposed to Hyperfilm™ ECL™ for 1-2 h.
ii) Chemifluorescence with ECF™: Hybridized blots are detected with ECF substrate. The fluorescent signal accumulates over time, low sensitivity applications giving results after one hour, high sensitivity applications usually requiring overnight incubation with substrate.
Detection time: 1-16 h.
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Product related documents
Application note
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. <para/>Hybridization time:15 min pre-hybridization 2-16 h hybridization Stringency washes:30 min'))
 Chemiluminescence with CDP-Star%21%22Hybridized blots are detected with CDP-Star reagent. Following a 2 min incubation, blots are exposed to <ExtText>Hyperfilm%21%22 ECL%21%22</ExtText><ExtRef>/aptrix/upp01077.nsf/Content/western_blot_site%5Cdetection%5Cproduct_selection_guide_autoradiography_films%5Chyperfilm_ecl?OpenDocument&parentid=25800095&moduleid=46995</ExtRef> for 1-2 h.<para/> ii) Chemifluorescence with ECF%21%22: Hybridized blots are detected with ECF substrate. The fluorescent signal accumulates over time, low sensitivity applications giving results after one hour, high sensitivity applications usually requiring overnight incubation with substrate.Detection time: 1-16 h.'))
