Project description:NPAC ChIP were performed by anti-Flag and anti-HA tandem affinity purification from HeLa stably expressing Flag-HA tagged NPAC from pOZ-N vector, and enrichement on chromosome 3, 21, and 22 were determined by chip microarray analysis using Affymatrix HumanTiling 2.0 arrays
Project description:SMYD3, a member of the SET and MYND domain-containing (SMYD) family, is a histone methyltransferase (HMT) and transcription factor that plays an important role in transcriptional regulation in human carcinogenesis.In an effort to better understanding the mechanistic role of SMYD3, affinity purification and mass spectrometry assays were used to identify proteins associated with SMYD3 in vivo. In these experiments, FLAG-tagged SMYD3 or an empty vector was stably expressed in LM3 cells. Cellular extracts were prepared and subjected to affinity purification using an anti-FLAG affinity gel. Immunocomplexed proteins were separated using SDS–PAGE and silver stained. Immunoprecipitated proteins in specific bands in comparison to the vector were gel extracted, trypsin digested and identified using liquid chromatography tandem mass spectrometry.
Project description:To clarify novel roles of IFITM3 during PDCoV infection,host cell proteins that interact with IFITM3 were screened by tandem affinity purification coupled with mass spectrometry (TAP/MS) in ST cell line stably expressing IFITM3 via lentivirus.
Project description:To identify the proteins interact with PDL1 protein in human cell lines, we analyzed the protein complexes using tandem affinity purification followed by mass spectrometry (TAP-MS) in HEK293T. We established the cell lines stably expressing PDL1 gene fused with Flag tags. We identified proteins associated with the bait in the isolated complexes using MS and searched the human databases for these proteins.
Project description:The protein interactome of the human C3orf33 protein was explored in U2OS cells through affinity purification-mass spectrometry (AP-MS). Specifically, U2OS cells stably expressing C3orf33 with either an N-terminus or a C-terminus 3XFlag tag were initially lysed using RIPA buffer. Subsequently, the proteins associated with C3orf33 were purified with anti-flag magnetic beads. Proteins were then eluted from the beads using competition with 3×Flag peptides and subsequently analyzed with mass spectrometry.
Project description:identify the potential partners of STC1 at the protein level, we performed mass spectrometry on B16-F10 tumor cells stably expressing FLAG-tagged STC1.
Project description:To elucidate the role of RNF4 in DNA replication and fork reversal, we performed tandem affinity purification (TAP) using a HEK293T cell line that stably expresses SFB-tagged(S-protein tag, Flag epitope tag, and streptavidin-binding peptide tag) wild-type and CS mutant to isolate proteins that associate with RNF4.
Project description:LC-MS/MS-based identification of HLA-peptides is poised to provide a deep understanding of the rules underlying antigen presentation. However, a key obstacle limiting the utility of MS data is the ambiguity arising from the co-expression of multiple HLA alleles. Here, we introduce a strategy for profiling the HLA ligandome one allele at a time. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing a novel spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of novel binding motifs, and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on datasets of peptides with measured affinities. We thus demonstrate a scalable strategy for systematically learning the rules of endogenous antigen presentation.
Project description:For a single step AP-MS identification of PME1 interactome was established a cell line stably expressing either Strep-tag alone or Strep_PME-1 fusion protein. After Strep-tag affinity purification, proteins were separated on SDS-PAGE gel, silver stained and protein bands that showed visually enrichment in StrepPME-1 samples compared to Strep-tag samples were isolated from both lanes for MS analysis.
Project description:We performed affinity purification of PRRX1 using cells expressing Flag tagged PRRX1 isoforms - PRRX1A or PRRX1B, and carried out mass spectrometry (AP-MS) analysis.