蛋白质相互作用高通量筛选和检测服务

人源蛋白质与文库相互作用的PCA筛选

由客户提供序列正确的基因克隆及其相关信息。如果诱饵蛋白基因克隆在本公司文库以内，则不需要提供。

周期：每个蛋白质与文库相互作用的PCA筛选实验周期为90个工作日。

服务完成时提交：

1). 技术服务报告（实验操作流程，数据、图表）

2). 与诱饵蛋白相互作用的蛋白列表。

人源蛋白质两两之间相互作用的PCA检测

由客户提供序列正确的基因克隆及其相关信息。如果基因克隆在本公司文库以内，则不需要提供。

周期：每对相互作用的PCA实验为60个工作日。

服务完成时提交：

1).技术服务报告（实验操作流程，实验数据、图表、照片）。

2).相互作用阳性克隆质粒实物。

利用细胞免疫荧光验证人源蛋白质的共定位

由客户提供序列正确的基因克隆及其相关信息。如果诱饵蛋白基因克隆在本公司文库以内，则不需要提供。如果需要内源抗体和指定的细胞系，请由客户提供。本公司提供Flag标签的表达质粒。

周期：每个蛋白的实验为60个工作日。

验证服务。

其他服务：提供HA, GST, Myc， GFP等质粒，表达标签蛋白的细胞系。

利用共沉淀验证人源蛋白质的相互作用

由客户提供序列正确的基因克隆及其相关信息。如果诱饵蛋白基因克隆在本公司文库以内，则不需要提供。如果需要内源抗体和指定的细胞系，请由客户提供。本公司提供Flag或GST标签的表达质粒。

周期：每对相互作用的实验为60个工作日。

验证服务。

其他服务：提供 HA, Myc， GFP等质粒，表达标签蛋白的细胞系。

服务完成时提交：

1).技术服务报告（实验操作流程，实验数据、图表、照片）。

2).相互作用阳性克隆质粒实物。

1. Chen Y, et al. (2015) Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly, trafficking and telomere maintenance. Nucleic acids research 43(1):385-395.

2. Han X, et al. (2013) Akt regulates TPP1 homodimerization and telomere protection. Aging cell 12(6):1091-1099.

3. Feng X, et al. (2013) The telomere-associated homeobox-containing protein TAH1/HMBOX1 participates in telomere maintenance in ALT cells. Journal of cell science 126(Pt 17):3982-3989.

4. Zheng ZY, et al. (2012) CHMP6 and VPS4A mediate the recycling of Ras to the plasma membrane to promote growth factor signaling. Oncogene 31(43):4630-4638.

5. Lee OH, et al. (2011) Genome-wide YFP fluorescence complementation screen identifies new regulators for telomere signaling in human cells. Molecular & cellular proteomics : MCP 10(2):M110 001628.

6. Wu W,  et al (2009) CARD9 facilitates microbe-elicited production of reactive oxygen species by regulating the LyGDI-Rac1 complex. Nature immunology 10(11):1208-1214.

7. Wang Z, et al. (2009) Presynaptic and postsynaptic interaction of the amyloid precursor protein promotes peripheral and central synaptogenesis. The Journal of neuroscience : the official journal of the Society for Neuroscience 29(35):10788-10801.

8. Kim H, et al. (2009) TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs. Nature structural & molecular biology 16(4):372-379.

9. Liang J, et al. (2008) Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells. Nature cell biology 10(6):731-739.

10. Chen LY, Liu D, & Songyang Z (2007) Telomere maintenance through spatial control of telomeric proteins. Molecular and cellular biology 27(16):5898-5909.

11. Ding Z, et al. (2006) A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proceedings of the National Academy of Sciences of the United States of America 103(41):15014-15019.

Ding Z et al. PNAS 2006;103:15014-15019

Pilot screen for AKT1-interaction partners.

Pilot screen for AKT1-interaction partners. (A) AKT1 interaction with PDK1 demonstrated by PCA. IFPN-AKT1 HeLa Tet-on cells stably transfected to coexpress PDK1-IFPC yielded highly fluorescent cells with enhanced fluorescence at the cell membrane and points of cell–cell contact. Inhibition of PI3K with LY294002 (20 μM) for 3 hr abrogated membrane localization. (B) Selected RePCA clones with different fluorescent patterns. Expanded clones were assessed for fluorescence after doxycycline induction. The target genes activated in each clone are shown under each image. (C) SSB clone showing nuclear fluorescence. (D) SLC3A2 clone showing membrane fluorescence

Zheng ZY, et al. Oncogene 2012 31(43):4630-4638.

Screen human Ras-interacting proteins in live mammalian cells using PCA

CHMP6 and VPS4A are novel H-Ras binding proteins. (a) HT1080 cells expressing Yn-H-Ras(12V) and either Yc-tagged CHMP6 or VPS4A, together with CFP-tagged GalT, Rab5A, Rab7A, or Rab11A, which mark Golgi, early (E.) endosomes, late (L.) endosomes/MVBs, or recycling (R.) endosomes, respectively, were analyzed by confocal microscopy. CFP and the reconstituted YFP signals were pseudo-colored red and green. Dotted lines mark the cell boundaries. Insets are scaled up images to better show co-localization between the CFP and YFP signals.

Wu W,  et al Nature immunology 2009 10(11):1208-1214.

Screening of CARD9 interacting proteins in responses to bacteria infection

Confocal microscopy of NYCD9 cells (a) and NYCD9-S18 cells (b) left unchallenged (None) or challenged for 30 min with heat-inactivated L. monocytogenes or E. coli, then fixed and made permeable, and then stained with rabbit antibody to LAMP2 (anti-LAMP2) plus Alex Fluor 594–labeled goat anti–rabbit immunoglobulin (red) and analyzed for the emission of YFP fluorescence (green). For each group of four images: top left, YFP fluorescence (pseudocolored green); top right, red fluorescence; bottom left, merged green and red fluorescence; bottom right, merged green and red fluorescence and transmission imaging. Original magnification, times63. Data are representative of two experiments.

Lee O et al. Mol Cell Proteomics 2011;10:M110.001628

Identification of proteins that associate with the six telomeric proteins through our PCA-based screens. 

A, Screening data for TRF2. On the basis of CytoArray analysis, a cutoff value of 0.090 (90% confidence level) for the weighted positive ratio (WPR) was applied (indicated by the red line). X axis, prey proteins scored in the screen. Y axis, WPR values. Arrows indicate known interacting prey proteins. B, A heat-map of the identified interaction partners of the core telomere associated proteins. C, Enriched biological processes identified by the screens.