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Protocol
Proximity ligation assay
Keywords: Proximity ligation assay, PLA, protein detection, protein-protein interaction, macromolecule interaction, protein characterization.
Proximity ligation assay is a protein-analyzing method for detection and characterization of proteins with high specificity and sensitivity.
Proximity ligation assay (PLA) is a recently developed method in which specific proteins are analyzed by converting detection reactions to DNA sequences. In this method target molecules are recognized by two or more proximity probes that are prepared by attaching DNA strands to affinity binders. When three of such probes bind to a common target molecule/molecules, the free ends of two of the probes are brought in proximity and are capable of hybridizing, at some distance from each other, to an oligonucleotide present on a third proximity probe. A cassette oligo, that precisely spans the gap between the 3’ and 5’ ends of the first two oligonucleotides, is added, allowing the ends to be joined by enzymatic DNA ligation. The ligation products are then amplified by PCR and distinguished from unreacted probes.
Required Materials
- PBS , Homemade
- ATP
- Real-time PCR
- Optical caps
- Optical tubes
- Biotinylated antibodies , from different companies depending on availability
- Streptavidin conjugated proximity arms
- poly adenylic acid
- Biotin
- BSA
- Platinum Taq DNA polymerase + buffer
Instructions
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Proximity probe conjugation (5Y53TC)
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Dilute your antibodies to 100nM in dilution buffer.
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Dilute the 3 arms to 100nM in dilution buffer.
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Mix equal volume of the biotinylated antibody/antibodies with the 3 proximity arms, respectably.
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Incubate at room temperature for one hour.
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The conjugates can be used immediately or can be stored at +4°C up to one month depending on the quality of the antibody.
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Sample preparation
Dilute the sample in proximity ligation (PLA) buffer; for instance a 10 times dilution series from 100nM to 0,01pM.
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Proximity probe mixture
Mix the three conjugates in PLA buffer to a concentration of 250pM each.
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Proximity ligation (5Y5HX2)
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Add 5µl of the proximity probe mixture to each optical PCR tube.
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Add 1µl of appropriate sample dilution to each tube.
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Cap the tubes and spin down the mixtures for 2-3 seconds.
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Incubate the mixtures at the temperature and the time appropriate for the antibodies. (If you are not sure about the kinetics for the antibodies, you van start with 1 hour at +37°C, and then optimize the temperature and the time).
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Ligation and PCR mixture
Prepare the ligation and PCR mixture for the appropriate number of reactions.
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PCR (5Y82JX)
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Before you open the tubes, spin the tubes for 3-4 seconds.
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Add 45µl of Ligation/PCR mixture to each tube, cap the tubes, and spin the tubes for 3-4 seconds.
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Incubate for 5 min at RT.
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Transfer the tubes to a real-time PCR device and start the program.
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PCR program
1x (95°C for 2 min)
45x (95°C for 15 sec, 60°C for 60 sec)
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Data analysis
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