TAQMAN PROTEIN ASSAYS - VHIR

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:
1 2 3 4 5 6
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 (*) PER REACTION (*)
MW96 287,45 € 2,99 €
MW384 1.074,53 € 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
(paqui.gallego@vhir.org, phone ext. 4179).