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Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

ABSTRACT: Data from ProteomeXchange, PXD ID: PXD002534. File: GR_130129_20_OT3_NT_L3MBTL2_FAB_31_G11_2_HEK293.mzXML. Published as part of Nat Methods. 2015 Jun 29 . From the Abstract: {{i}} ... We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1, 124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins ... {{/i}}

INSTRUMENT(S): Instrument

ORGANISM(S): Homo_sapiens_viruses, Human_female

DISEASE(S): Not Available

SUBMITTER: Marcon E, et al.

PROVIDER: GPM32320016573 | GPMDB |

REPOSITORIES: GPMDB

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Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Marcon Edyta E Jain Harshika H Bhattacharya Anandi A Guo Hongbo H Phanse Sadhna S Pu Shuye S Byram Gregory G Collins Ben C BC Dowdell Evan E Fenner Maria M Guo Xinghua X Hutchinson Ashley A Kennedy Jacob J JJ Krastins Bryan B Larsen Brett B Lin Zhen-Yuan ZY Lopez Mary F MF Loppnau Peter P Miersch Shane S Nguyen Tin T Olsen Jonathan B JB Paduch Marcin M Ravichandran Mani M Seitova Alma A Vadali Gouri G Vogelsang Maryann S MS Whiteaker Jeffrey R JR Zhong Guoqing G Zhong Nan N Zhao Lei L Aebersold Ruedi R Arrowsmith Cheryl H CH Emili Andrew A Frappier Lori L Gingras Anne-Claude AC Gstaiger Matthias M Paulovich Amanda G AG Koide Shohei S Kossiakoff Anthony A AA Sidhu Sachdev S SS Wodak Shoshana J SJ Gräslund Susanne S Greenblatt Jack F JF Edwards Aled M AM

Nature methods 20150629 8

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors fro ...[more]

PMID: 26121405

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Project description:Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem-cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila Kc cells. We found that Pc associates with large genomic regions of up to ~150kb in size, hereafter referred to as âPc-domainsâ. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 lysine 27 and in general transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains. Keywords: DamID, Chromatin immunoprecipitation, ChIP-chip To study PcG binding profiles we used DamID, which is based on the ability of a chromatin protein fused to E.coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000).

Dataset Information

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Publications

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

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