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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

Comparing standard NIR detection and AMSA. (a) Western blot specificity of TSA and AMSA. Detection of PSA (star-symbol) using standard NIR procedure (scan intensity 5), antibody-mediated signal amplification (scan intensity 2.5) and TSA (scan intensity 2.5). Each lane corresponds to loading 5 μg total protein from prostate cancer tissue or PSA-free negative controls: Colon cancer cell lines HT29 (1), SW480 (2), mouse liver lysate (3), and prostate cancer tissue (4). (b) Western blot analysis of 14 proteins in seven human cancer cell lines with standard NIR detection. (c) Samples were analyzed by RPPA to compare standard NIR detection and AMSA using the same set of antibodies as shown for the Western blot. The correlation analyses were based on log signal intensities.
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Figure 3: Comparing standard NIR detection and AMSA. (a) Western blot specificity of TSA and AMSA. Detection of PSA (star-symbol) using standard NIR procedure (scan intensity 5), antibody-mediated signal amplification (scan intensity 2.5) and TSA (scan intensity 2.5). Each lane corresponds to loading 5 μg total protein from prostate cancer tissue or PSA-free negative controls: Colon cancer cell lines HT29 (1), SW480 (2), mouse liver lysate (3), and prostate cancer tissue (4). (b) Western blot analysis of 14 proteins in seven human cancer cell lines with standard NIR detection. (c) Samples were analyzed by RPPA to compare standard NIR detection and AMSA using the same set of antibodies as shown for the Western blot. The correlation analyses were based on log signal intensities.

Mentions: The specificity of the AMSA approach was validated by detecting PSA in different samples on Western blots (Figure 3a) and compared to blots detected in parallel by standard NIR and TSA-based signal amplification. In detail, the presence of PSA was probed in a human prostate cancer lysate and in samples serving as PSA-free negative controls such as human colon cancer cell lines (HT29, SW480) or homogenized mouse liver. Strong and specific PSA signals were detected only in prostate cancer samples and when using standard NIR detection and AMSA. The TSA detection yielded also a strong PSA band on Western Blots. Nonetheless, several additional bands were observerd in prostate tumor samples as well as for the mouse liver lysates. To sum up, unspecific bands were not observed in cell line samples and when using standard NIR detection or AMSA. Thus, the numerous additional bands emerging during the TSA procedure were also observed when the anti-PSA antibody was omitted (data not shown) and possibly resulted from insufficient masking of biotin proteins as artifact of the TSA detection.


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)

Comparing standard NIR detection and AMSA. (a) Western blot specificity of TSA and AMSA. Detection of PSA (star-symbol) using standard NIR procedure (scan intensity 5), antibody-mediated signal amplification (scan intensity 2.5) and TSA (scan intensity 2.5). Each lane corresponds to loading 5 μg total protein from prostate cancer tissue or PSA-free negative controls: Colon cancer cell lines HT29 (1), SW480 (2), mouse liver lysate (3), and prostate cancer tissue (4). (b) Western blot analysis of 14 proteins in seven human cancer cell lines with standard NIR detection. (c) Samples were analyzed by RPPA to compare standard NIR detection and AMSA using the same set of antibodies as shown for the Western blot. The correlation analyses were based on log signal intensities.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Comparing standard NIR detection and AMSA. (a) Western blot specificity of TSA and AMSA. Detection of PSA (star-symbol) using standard NIR procedure (scan intensity 5), antibody-mediated signal amplification (scan intensity 2.5) and TSA (scan intensity 2.5). Each lane corresponds to loading 5 μg total protein from prostate cancer tissue or PSA-free negative controls: Colon cancer cell lines HT29 (1), SW480 (2), mouse liver lysate (3), and prostate cancer tissue (4). (b) Western blot analysis of 14 proteins in seven human cancer cell lines with standard NIR detection. (c) Samples were analyzed by RPPA to compare standard NIR detection and AMSA using the same set of antibodies as shown for the Western blot. The correlation analyses were based on log signal intensities.
Mentions: The specificity of the AMSA approach was validated by detecting PSA in different samples on Western blots (Figure 3a) and compared to blots detected in parallel by standard NIR and TSA-based signal amplification. In detail, the presence of PSA was probed in a human prostate cancer lysate and in samples serving as PSA-free negative controls such as human colon cancer cell lines (HT29, SW480) or homogenized mouse liver. Strong and specific PSA signals were detected only in prostate cancer samples and when using standard NIR detection and AMSA. The TSA detection yielded also a strong PSA band on Western Blots. Nonetheless, several additional bands were observerd in prostate tumor samples as well as for the mouse liver lysates. To sum up, unspecific bands were not observed in cell line samples and when using standard NIR detection or AMSA. Thus, the numerous additional bands emerging during the TSA procedure were also observed when the anti-PSA antibody was omitted (data not shown) and possibly resulted from insufficient masking of biotin proteins as artifact of the TSA detection.

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