Download taqman protein assays

TAQMAN PROTEIN ASSAYS 1. INTRODUCTION. This technology is based in the Taqman technology applied to protein detection and quantification. The protocol uses biotinylated antibodies, which are ligated to two streptavidin‐conjugated oligonucleotides that contain a complementary region. When the two conjugated antibodies bind to an epitope and are in close proximity, the oligonucleotides can be ligated, serving as the template for real‐time PCR amplification and quantification. Ligated oligos are subsequently used as the binding site for the Taqman probe to perform the qPCR reaction, which generates a specific signal for the protein‐antibody complexes. 2. METHOD DESCRIPTION. The protocol is quite simple and can be performed using the real‐time qPCR instruments available at the UAT. Protocol steps: 4
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1 1) Cell lysis and construction of serial dilutions. 2) Biotin‐streptavidin mediated interaction of the biotinylated antibodies with the oligonucleotides. 3) Incubation of the cell lysates with the Taqman protein assay probes (antibody+oligo). 4) Hybridization of the assay probe oligonucleotides using the “connector” oligo allowing further ligation. Ligase inactivation. 5) qPCR reaction. 6) Data analysis. 3. ADVANTAGES.  High sensitivity: it uses 10 to 50‐fold less input sample than other traditional protein analysis methods. The typical input range is 10‐500 cells or tissue lysate containing 1‐100 ng total protein. 
Relative quantification: data normalization referred to initial cell number or total protein input can be done (suitable endogenous controls do no yet exist). Fold change comparison between samples is also possible. 
This technique allows to develop new applications that can´t be done using traditional protein analysis techniques, for example, protein, mRNA and miRNA can be analyzed on the same sample, which is of high biological potential. The effect of interfering RNAs on protein expression can be easily monitorized. Protein interaction “in vitro” can be investigated using two different antibodies.  TaqMan protein expression assays are faster and use significantly less input material than western blotting.  Sample analysis format is 96 or 384 well‐plate, so many samples can be processed at the same time. 4. DISADVANTAGES.  Limited disponibility of comercial biotinylated antibodies (sometimes they will need to be hand‐made).  Antibodies must be validated before starting the assay (this is also true for western blotting).  Manual set‐up of multiwell plates is a tedious work and can introduce pipetting errors and variability. 5. WORKFLOW. Cell lysates and initial cell counting/total input protein quantification have to be done by the researcher. The UAT has to be provided with the cell lysate and the biotinylated antibodies. The UAT technician in charge will perform the rest of the protocol, including preliminary data analysis of the qPCR results. 6. SERVICE RATES. Life Technologies is currently offering us a free trial to validate the biotinylated antibodies in a cell lysate. The UAT offers a promotional price meanwhile the technique is being optimized: PLATE FORMAT PER PLATE (*)
MW96 287,45 € MW384 1.074,53 € PER REACTION (*) 2,99 € 2,80 € (*) It includes sample processing, consumables, qPCR machine utilization and preliminary qPCR data quality control and analysis. To start offering this service a minimum number of three VHIR research groups is required. For more information please contact the technician in charge, Paqui Gallego ([email protected], phone ext. 4179).