RNAi-based validation of antibodies for reverse phase protein arrays
<p>Abstract</p> <p>Background</p> <p>Reverse phase protein arrays (RPPA) have been demonstrated to be a useful experimental platform for quantitative protein profiling in a high-throughput format. Target protein detection relies on the readout obtained from a single det...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2010-12-01
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| Series: | Proteome Science |
| Online Access: | http://www.proteomesci.com/content/8/1/69 |
| _version_ | 1828290037295349760 |
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| author | Sahin Özgür Wiemann Stefan Schmidt Christian Uhlmann Stefan Mannsperger Heiko A Korf Ulrike |
| author_facet | Sahin Özgür Wiemann Stefan Schmidt Christian Uhlmann Stefan Mannsperger Heiko A Korf Ulrike |
| author_sort | Sahin Özgür |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Reverse phase protein arrays (RPPA) have been demonstrated to be a useful experimental platform for quantitative protein profiling in a high-throughput format. Target protein detection relies on the readout obtained from a single detection antibody. For this reason, antibody specificity is a key factor for RPPA. RNAi allows the specific knockdown of a target protein in complex samples and was therefore examined for its utility to assess antibody performance for RPPA applications.</p> <p>Results</p> <p>To proof the feasibility of our strategy, two different anti-EGFR antibodies were compared by RPPA. Both detected the knockdown of EGFR but at a different rate. Western blot data were used to identify the most reliable antibody. The RNAi approach was also used to characterize commercial anti-STAT3 antibodies. Out of ten tested anti-STAT3 antibodies, four antibodies detected the STAT3-knockdown at 80-85%, and the most sensitive anti-STAT3 antibody was identified by comparing detection limits. Thus, the use of RNAi for RPPA antibody validation was demonstrated to be a stringent approach to identify highly specific and highly sensitive antibodies. Furthermore, the RNAi/RPPA strategy is also useful for the validation of isoform-specific antibodies as shown for the identification of AKT1/AKT2 and CCND1/CCND3-specific antibodies.</p> <p>Conclusions</p> <p>RNAi is a valuable tool for the identification of very specific and highly sensitive antibodies, and is therefore especially useful for the validation of RPPA-suitable detection antibodies. On the other hand, when a set of well-characterized RPPA-antibodies is available, large-scale RNAi experiments analyzed by RPPA might deliver useful information for network reconstruction.</p> |
| first_indexed | 2024-04-13T10:30:43Z |
| format | Article |
| id | doaj.art-87922132c039457bb17b6520a731e25e |
| institution | Directory Open Access Journal |
| issn | 1477-5956 |
| language | English |
| last_indexed | 2024-04-13T10:30:43Z |
| publishDate | 2010-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Proteome Science |
| spelling | doaj.art-87922132c039457bb17b6520a731e25e2022-12-22T02:50:11ZengBMCProteome Science1477-59562010-12-01816910.1186/1477-5956-8-69RNAi-based validation of antibodies for reverse phase protein arraysSahin ÖzgürWiemann StefanSchmidt ChristianUhlmann StefanMannsperger Heiko AKorf Ulrike<p>Abstract</p> <p>Background</p> <p>Reverse phase protein arrays (RPPA) have been demonstrated to be a useful experimental platform for quantitative protein profiling in a high-throughput format. Target protein detection relies on the readout obtained from a single detection antibody. For this reason, antibody specificity is a key factor for RPPA. RNAi allows the specific knockdown of a target protein in complex samples and was therefore examined for its utility to assess antibody performance for RPPA applications.</p> <p>Results</p> <p>To proof the feasibility of our strategy, two different anti-EGFR antibodies were compared by RPPA. Both detected the knockdown of EGFR but at a different rate. Western blot data were used to identify the most reliable antibody. The RNAi approach was also used to characterize commercial anti-STAT3 antibodies. Out of ten tested anti-STAT3 antibodies, four antibodies detected the STAT3-knockdown at 80-85%, and the most sensitive anti-STAT3 antibody was identified by comparing detection limits. Thus, the use of RNAi for RPPA antibody validation was demonstrated to be a stringent approach to identify highly specific and highly sensitive antibodies. Furthermore, the RNAi/RPPA strategy is also useful for the validation of isoform-specific antibodies as shown for the identification of AKT1/AKT2 and CCND1/CCND3-specific antibodies.</p> <p>Conclusions</p> <p>RNAi is a valuable tool for the identification of very specific and highly sensitive antibodies, and is therefore especially useful for the validation of RPPA-suitable detection antibodies. On the other hand, when a set of well-characterized RPPA-antibodies is available, large-scale RNAi experiments analyzed by RPPA might deliver useful information for network reconstruction.</p>http://www.proteomesci.com/content/8/1/69 |
| spellingShingle | Sahin Özgür Wiemann Stefan Schmidt Christian Uhlmann Stefan Mannsperger Heiko A Korf Ulrike RNAi-based validation of antibodies for reverse phase protein arrays Proteome Science |
| title | RNAi-based validation of antibodies for reverse phase protein arrays |
| title_full | RNAi-based validation of antibodies for reverse phase protein arrays |
| title_fullStr | RNAi-based validation of antibodies for reverse phase protein arrays |
| title_full_unstemmed | RNAi-based validation of antibodies for reverse phase protein arrays |
| title_short | RNAi-based validation of antibodies for reverse phase protein arrays |
| title_sort | rnai based validation of antibodies for reverse phase protein arrays |
| url | http://www.proteomesci.com/content/8/1/69 |
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