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Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification.

Brase JC, Mannsperger H, Fröhlich H, Gade S, Schmidt C, Wiemann S, Beissbarth T, Schlomm T, Sültmann H, Korf U - Proteome Sci (2010)

Bottom Line: The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection.Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs.Contrasting other amplification approaches it allows target protein detection over a large linear range.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany. u.korf@dkfz.de.

ABSTRACT

Background: Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level.

Results: A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins.

Conclusions: Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range.

No MeSH data available.


Related in: MedlinePlus

Optimizing number of amplification cycles. Twelve replicate spots of a colon cancer cell line containing 35 fg pERK were printed on nitrocellulose-coated slides. Detection of pERK was performed by standard near-infrared detection and AMSA comprising different rounds of amplification. Median fluorescence (black) and signal-to-noise ratios (red) were calculated. The blue line summarizes the results for AMSA optimization.
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Figure 2: Optimizing number of amplification cycles. Twelve replicate spots of a colon cancer cell line containing 35 fg pERK were printed on nitrocellulose-coated slides. Detection of pERK was performed by standard near-infrared detection and AMSA comprising different rounds of amplification. Median fluorescence (black) and signal-to-noise ratios (red) were calculated. The blue line summarizes the results for AMSA optimization.

Mentions: As a side-effect of signal amplification an increase of background signal was supposed. Therefore, the AMSA-dependent background signal gain was quantified using a spike-in of phosphorylated recombinant ERK1. This way, the best trade-off between specific and background signal intensity gain was assessed. RPPA signal detection was based on standard NIR or AMSA with one to five rounds of signal amplification (Figure 2). Thus, background signal intensity gain correlated with the number of amplification steps performed. After the first two cycles of amplification signal-to-noise ratios were not improved compared to those obtained by standard NIR detection. However, after three amplification cycles the signal-to-noise ratio started to increase and the best result was obtained by carrying out four amplification cycles and this number of cycles was consequently chosen as basis for the AMSA procedure.


Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification.

Brase JC, Mannsperger H, Fröhlich H, Gade S, Schmidt C, Wiemann S, Beissbarth T, Schlomm T, Sültmann H, Korf U - Proteome Sci (2010)

Optimizing number of amplification cycles. Twelve replicate spots of a colon cancer cell line containing 35 fg pERK were printed on nitrocellulose-coated slides. Detection of pERK was performed by standard near-infrared detection and AMSA comprising different rounds of amplification. Median fluorescence (black) and signal-to-noise ratios (red) were calculated. The blue line summarizes the results for AMSA optimization.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908584&req=5

Figure 2: Optimizing number of amplification cycles. Twelve replicate spots of a colon cancer cell line containing 35 fg pERK were printed on nitrocellulose-coated slides. Detection of pERK was performed by standard near-infrared detection and AMSA comprising different rounds of amplification. Median fluorescence (black) and signal-to-noise ratios (red) were calculated. The blue line summarizes the results for AMSA optimization.
Mentions: As a side-effect of signal amplification an increase of background signal was supposed. Therefore, the AMSA-dependent background signal gain was quantified using a spike-in of phosphorylated recombinant ERK1. This way, the best trade-off between specific and background signal intensity gain was assessed. RPPA signal detection was based on standard NIR or AMSA with one to five rounds of signal amplification (Figure 2). Thus, background signal intensity gain correlated with the number of amplification steps performed. After the first two cycles of amplification signal-to-noise ratios were not improved compared to those obtained by standard NIR detection. However, after three amplification cycles the signal-to-noise ratio started to increase and the best result was obtained by carrying out four amplification cycles and this number of cycles was consequently chosen as basis for the AMSA procedure.

Bottom Line: The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection.Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs.Contrasting other amplification approaches it allows target protein detection over a large linear range.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany. u.korf@dkfz.de.

ABSTRACT

Background: Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level.

Results: A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins.

Conclusions: Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range.

No MeSH data available.


Related in: MedlinePlus