Journal: Journal of Assisted Reproduction and Genetics

Article Title: Methodological approaches in 16S sequencing of female reproductive tract in fertility patients: a review

doi: 10.1007/s10815-024-03292-6

Figure Lengend Snippet: Summary of all included articles identified highlighting the methodologies and key microorganisms identified within each study

Article Snippet: Komiya et al. [ ] , Vagina , Vaginal lavage and swabs , Lavage samples stored at − 30 °C. Swabs stored at room temperature following a preservative liquid , DNA extracted via QIAamp UCP Pathogen Mini Kit (QIAGEN) , V1-V9, V3-V4 regions of the 16S rRNA gene via metagenomics using S-D-Bact-0008-c-S-20, 341F & 806R primers with anchor sequences on each , Nanopore , GUPPY/Minimap2 , Lactobacillus , Streptococcus , Atopobium , and Staphylococcus.

Techniques: Sampling, DNA Extraction, Sequencing, Extraction, Lysis, DNA Purification, Incubation, Software, Preserving, Transferring, Isolation

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Methodological approaches in 16S sequencing of female reproductive tract in fertility patients: a review

doi: 10.1007/s10815-024-03292-6

Figure Lengend Snippet: Comparison of different DNA extraction techniques key steps, advantages, and limitations [ 39 , 41 , 43 , 53 ]

Article Snippet: Komiya et al. [ ] , Vagina , Vaginal lavage and swabs , Lavage samples stored at − 30 °C. Swabs stored at room temperature following a preservative liquid , DNA extracted via QIAamp UCP Pathogen Mini Kit (QIAGEN) , V1-V9, V3-V4 regions of the 16S rRNA gene via metagenomics using S-D-Bact-0008-c-S-20, 341F & 806R primers with anchor sequences on each , Nanopore , GUPPY/Minimap2 , Lactobacillus , Streptococcus , Atopobium , and Staphylococcus.

Techniques: Comparison, DNA Extraction, Extraction, Homogenization, Purification, Binding Assay, Isolation, Lysis, Concentration Assay, Bacteria, Membrane

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: FGF19/FGFR4 signaling axis confines and switches the role of melatonin in head and neck cancer metastasis

doi: 10.1186/s13046-021-01888-9

Figure Lengend Snippet: MT induces ER stress-associated upregulation of FGF19 in HNSCC cells. a The inhibitory effect of MT on cell viability determined after three-day treatment. b The inducible effect of MT on apoptosis determined by Annexin V FITC/PI staining after two-day treatment. Representative results and quantitative data from three independent experiments are shown in the left and right panels, respectively. c The effect of MT on the cleavage of Caspase 3 and Caspase 12 determined after two-day treatment. d The level changes in ER stress markers within the first 12 h following MT treatment. e The activation of elF2α-ATF4 signaling within the first 12 h following MT treatment. f The activation of FGF19-FGFR4 axis within the first 12 h following MT treatment. g Increased FGF19 mRNA expression within the first 12 h following MT treatment. * p < 0.05; ** p < 0.01

Article Snippet: The Melatonin ELISA Kit (BioVision, Milpitas, CA) and the Human FGF19 Quantikine® ELISA kit (R&D Systems, Minneapolis, MN) were used for quantitative measurement of MT and FGF19 in blood samples.

Techniques: Staining, Activation Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: FGF19/FGFR4 signaling axis confines and switches the role of melatonin in head and neck cancer metastasis

doi: 10.1186/s13046-021-01888-9

Figure Lengend Snippet: High-dose MT induces upregulation of FGF19 in HNSCC cells via ATF4. a The effects of high and low dose of MT on the activation of elF2α-ATF4-FGF19 signaling pathway in the indicated cell lines following MT treatment for 12 h. b MTNR1A mRNA levels in HNSCC cell lines and normal oral keratinocytes (hTERT). c , d The effects of high and low dose of MT on the increase of FGF19 levels in the orthotopic mouse tongue tumor model. The xenografts were harvested for western blotting analysis ( c ) after homogenization and IHC with the anti-FGF19 antibody ( d ). In ( d ), arrows indicate FGF19-positive tumor cells in the HN12-derived tumor xenografts. e The protein levels of FGF19 in human primary HNSCC tissues with high or low levels of serum MT determined by IHC. In ( d , e ), quantitative data are shown in the right panels. f , g The effect of ATF4 knockdown on FGF19 expression in HN12 cells at either protein ( f ) or mRNA levels ( g ). h The binding of ATF4 on the FGF19 gene promoter determined by ChIP-qPCR assay. A putative ATF4 binding site (AARE) on the FGF19 gene promoter is shown in the cartoon (above). i The enrichment of ATF4 on the FGF19 gene promoter determined by ChIP-qPCR assay in the presence or absence of 5 mM MT. * p < 0.05; ** p < 0.01

Article Snippet: The Melatonin ELISA Kit (BioVision, Milpitas, CA) and the Human FGF19 Quantikine® ELISA kit (R&D Systems, Minneapolis, MN) were used for quantitative measurement of MT and FGF19 in blood samples.

Techniques: Activation Assay, Western Blot, Homogenization, Derivative Assay, Knockdown, Expressing, Binding Assay, ChIP-qPCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: FGF19/FGFR4 signaling axis confines and switches the role of melatonin in head and neck cancer metastasis

doi: 10.1186/s13046-021-01888-9

Figure Lengend Snippet: FGF19-FGFR4 signaling axis confines the role of MT in suppressing cell motility via upregulating Vimentin in HNSCC cells. a The effect of MT at low and high doses on cell migration determined by wound healing assays within 16 h following treatment. b The effect of MT on Vimentin protein levels in HN12 cells determined by Western blotting. c The effect of FGF19 knockdown (shFGF19–1 and shFGF19–2) on Vimentin protein levels in HN12 cells determined by Western blotting. d The effect of MT on cell migration in HN12 cells on Vimentin protein levels with or without FGF19 knockdown (shFGF19 = shFGF19–1). e The effect of FGFR4 KO on Vimentin protein levels in HN12 cells determined by Western blotting. f The effect of 5 mM MT on cell migration in HN12 cells with or without the genetic inhibition of FGF19 (shFGF19) or FGFR4 (FGFR4 KO) gene expression. g , h The effect of MT on Vimentin levels ( g ) and cell migration ( h ) in HN12 cells in the presence or absence of FGFR4 inhibitor H3B-6527. In ( a , f , h ), representative images of cell migration and quantitative data from three independent experiments are shown in the left and right panels, respectively. * p < 0.05; ** p < 0.01

Article Snippet: The Melatonin ELISA Kit (BioVision, Milpitas, CA) and the Human FGF19 Quantikine® ELISA kit (R&D Systems, Minneapolis, MN) were used for quantitative measurement of MT and FGF19 in blood samples.

Techniques: Migration, Western Blot, Knockdown, Inhibition, Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: FGF19/FGFR4 signaling axis confines and switches the role of melatonin in head and neck cancer metastasis

doi: 10.1186/s13046-021-01888-9

Figure Lengend Snippet: FGF19-FGFR4 signaling axis switches the role of MT in cell motility upon long-term exposure to high-dose MT. a Increased levels of ATF4, FGF19, and Vimentin in MT-SE and MT-LE HN30 cells. b The increased ATF4 amount on the FGF19 gene promoter in MT-LE HN30 cells than MT-SE HN30 cells determined by ChIP-qPCR. c The morphological changes between MT-LE and its parental HN30 cells photographed under an inverted phase-contrast microscopy. d Comparison of migration capacity between MT-LE and its parental HN30 cells by wound healing assays. e Comparison of ex vivo invasion potential between MT-LE and its parental HN30 cells by SeedEZ™ 3D rings. f , g The effect of FGF19 knockdown (shFGF19) on cell migration ( f ) and Vimentin protein levels ( g ) in MT-LE HN30 cells. h , i The effect of H3B-6527 on cell migration ( h ) and Vimentin protein levels ( i ) in MT-LE HN30 cells. In ( d , e , f , h ) representative images and quantitative data from three independent experiments are shown in the left and right panels, respectively. MT-SE, HN30 cells under 12-h exposure of MT; MT-LE, HN30 cells under long-term exposure of MT. * p < 0.05; ** p < 0.01

Article Snippet: The Melatonin ELISA Kit (BioVision, Milpitas, CA) and the Human FGF19 Quantikine® ELISA kit (R&D Systems, Minneapolis, MN) were used for quantitative measurement of MT and FGF19 in blood samples.

Techniques: ChIP-qPCR, Microscopy, Comparison, Migration, Ex Vivo, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: FGF19/FGFR4 signaling axis confines and switches the role of melatonin in head and neck cancer metastasis

doi: 10.1186/s13046-021-01888-9

Figure Lengend Snippet: Knockdown of FGF19 attenuates the LNMets of MT-LE HNSCC cells in orthotopic mice of tongue tumors. a , b Bioluminescence images of tongue tumors ( a ) and tongue tumor weight ( b ) in mice implanted with the indicated cells after a four-week inoculation. c Bioluminescence images of tumor dissemination in lymph nodes in mice implanted with the indicated cells after a four-week inoculation. d The protein levels of Vimentin in tumor xenografts from mice implanted with the indicated cells determined by IHC. In ( a , c , d ), representative images and quantitative data are shown in the left and right panels, respectively. * p < 0.05; ** p < 0.01

Article Snippet: The Melatonin ELISA Kit (BioVision, Milpitas, CA) and the Human FGF19 Quantikine® ELISA kit (R&D Systems, Minneapolis, MN) were used for quantitative measurement of MT and FGF19 in blood samples.

Techniques: Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: FGF19/FGFR4 signaling axis confines and switches the role of melatonin in head and neck cancer metastasis

doi: 10.1186/s13046-021-01888-9

Figure Lengend Snippet: Schematic representation of MT-mediated FGF19 signaling in HNSCC cells. High-dose MT has the potential to upregulate FGF19 gene expression through activating ER stress-responsive PERK-eIF2α-ATF4 signaling in HNSCC cells. Increased FGF19 is secreted and then recruited back to cells by its specific receptor FGFR4 and further activates Vimentin signaling, which in turn confines the role of MT in anti-metastasis

Article Snippet: The Melatonin ELISA Kit (BioVision, Milpitas, CA) and the Human FGF19 Quantikine® ELISA kit (R&D Systems, Minneapolis, MN) were used for quantitative measurement of MT and FGF19 in blood samples.

Techniques: Gene Expression

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Advanced Technology for Gene Delivery with Homing Peptides to Spinal Cord through Systemic Circulation in Mice

doi: 10.1016/j.omtm.2019.04.008

Figure Lengend Snippet: Gene Therapy for Neuropathic Pain by IL-4 Expression with SP1 Homing Peptides to the Spinal Cord (A) Scheme of SP1 peptide structure and binding of peptides with vectors. (B) Mechanical threshold of the hind paw before and after LPS injection with gene therapy in pBOS-IL4-YFP (IL-4, n = 7) and SP1 + pBOS-IL-4-YFP (SP1 + IL-4, n = 8) groups. (C) mRNA expression of IL-4 in the spinal cord after gene therapy by pBOS-YFP (CTR, n = 5), pBOS-IL4-YFP (IL-4, n = 5), or SP1 + pBOS-IL-4-YFP (SP1 + IL-4, n = 5). IL-4 mRNA expressions were standardized by GAPDH mRNA expression, and their relative expression ratios were calculated against the CTR group. (D) Immunohistochemistry of IL-4 in the spinal cord after gene therapy. Top shows YFP (green) and bottom shows IL-4 staining (blue). Scale bars, 100 μm. (E) IL-4 protein expression by ELISA analysis in spinal cord homogenized in PBS or RIPA buffer after the injection of pBOS-YFP (CTR, n = 5), pBOS-IL-4-YFP (IL-4, n = 5), or SP1 + pBOS-IL-4-YFP (SP1 + IL-4, n = 5). IL-4 protein contents were standardized by spinal cord protein weight. *p < 0.05 and **p < 0.01 versus others; # p < 0.01 in (E).

Article Snippet: The protein expression of IL-4 in each spinal cord was measured using a mouse IL-4 ELISA kit (R&D Systems, Minneapolis, MN, USA) after homogenization in PBS or RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1.0% NP-40) containing protease inhibitor (Sigma-Aldrich) to evaluate the secreted or intracellular IL-4.

Techniques: Expressing, Binding Assay, Injection, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Macromolecules

Article Title: Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

doi: 10.1016/j.ijbiomac.2022.12.251

Figure Lengend Snippet: Physicochemical properties of royal jelly (RJ)-PF 50 protein composites and genomic organization of the SARS-CoV-2 S protein. (a, b) 3D structures of MRJP2 (orange) and MRJP2 isoform X1 (yellow), respectively, were generated with the Iterative Threading ASSEmbly Refinement (I-TASSER) protein-modelling online server ( http://zhanglab.ccmb.med.umich.edu/I-TASSER/ ). (c) SDS-PAGE (12 %) chromatogram profile of RJ proteins stained with silver nitrate. Lane M: protein markers (M) with various molecular weights, Lane 1: PF 50 (RJ protein fraction precipitated at 40–50 % ammonium sulphate saturation), Lane 2: MRJP2 isoform X1, and Lane 3: MRJP2. MRJP2 and its isoform X1 were purified using CM-Sephadex ion-exchange column chromatography. (d) Predicted exposed and buried amino acid residues in MRJP2 and its isoform X1 using the DeepREx-WS webserver ( https://deeprex.biocomp.unibo.it ). The light orange highlighted regions represent the polar amino acid content of the two tested MRJPs. (e) Schematic representation of SARS-CoV-2 S glycoprotein, illustrating S1 and S2 subunits and showing the S1-receptor binding mechanism. The SARS-CoV-2 trimeric S glycoprotein crystallographic structure (PDB ID: 6VXX ) shows the fusion protein (FP), N-terminal domain (NTD), intracellular tail (I.T.), and transmembrane anchor (T.A.) protein domains, as well as the receptor-binding domain (RBD) that represents the human ACE2 receptor binding site. The box displays the genomic architecture of the S gene, highlighting the coding regions of the S1 and S2 subunits. (f) Schematic representation of the key mutations in the SARS-CoV-2 S protein of the UK (Alpha, B.1.1.7) and South African (Beta, B.1.351) variants of concern. These mutation sites were retrieved from the PDBsum database ( http://www.ebi.ac.uk/thornton-srv/databases/pdbsum ).

Article Snippet: In addition, Thaw Medium 1 (#60187, growth medium without puromycin), anti-S antibody (#101024; clone No. C-A11), luciferase (Luc) reagent (#60690), and SARS-CoV-2 RdRp homogeneous assay kit (#78109) were supplied from BPS Bioscience.

Techniques: Generated, SDS Page, Staining, Purification, Column Chromatography, Binding Assay, Mutagenesis

Journal: International Journal of Biological Macromolecules

Article Title: Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

doi: 10.1016/j.ijbiomac.2022.12.251

Figure Lengend Snippet: PF 50 (MRJP2 with its isoform X1) neutralizes SARS-CoV-2 UK S protein (S UK )-mediated pseudovirus entry and structures models. (a, c) Surface and cartoon representations of trimeric SARS CoV-2 S UK (PDB ID: 7LWT , S1 “green “, RBD “dark red”, S2 “pale purple”) with multivalent interactions of three MRJP2 and MRJP2 X1 molecules (orange and yellow colored cartoons, respectively), followed by the human ACE2 (PDB ID: 1R42 “purple”). The two views of the docking model (a, c) are rotated by 180°. (b, d) The interacting amino acid residues (black) between SARS-CoV-2 S UK -RBD (only the matching residues with S UK RBD-ACE2 complex's interface pocket were presented) and the three molecules of (b) MRJP2 (orange) and one molecule of (d) MRJP2 X1 (yellow). The yellow-highlighted residues are those responsible for the high virulence and transmissibility of the UK SARS-CoV-2. The docked complex structure (a) was given by the PatchDock server ( https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php ) and was visualized by Discovery Studio 2020 Client software. (e) The binding affinity (ΔG) and other PDBePISA ( https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver ) results (hydrogen bonds “HB”, area, and residues number “ i N res ”) for the interfaces of the docked complexes obtained. The pseudotyped SARS-CoV-2 S UK was preincubated for 1 h with a three-fold serially diluted (f) PF 50 or (g) S1 neutralizing antibody (clone No. C-A11), and then the S UK /PF 50 mixture was incubated for 48 h with ACE2-HEK 293 T cells. A luciferase assay system was utilized to evaluate the neutralization potencies of tested proteins. The half-maximal inhibitory concentration (IC 50 ) values were determined using GraphPad Prism version 9.2.0 by fitting the luciferase activity from serially diluted PF 50 or antibody to a sigmoidal dose-response curve. (f) The cytotoxicity (red line) of PF 50 on the ACE2-HEK 293 T cells was done using the cell titer-Glo luminescent cell viability assay. Data ( n = 3) is represented as the mean ± S.E.

Article Snippet: In addition, Thaw Medium 1 (#60187, growth medium without puromycin), anti-S antibody (#101024; clone No. C-A11), luciferase (Luc) reagent (#60690), and SARS-CoV-2 RdRp homogeneous assay kit (#78109) were supplied from BPS Bioscience.

Techniques: Software, Binding Assay, Incubation, Luciferase, Neutralization, Concentration Assay, Activity Assay, Cell Viability Assay

Journal: International Journal of Biological Macromolecules

Article Title: Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

doi: 10.1016/j.ijbiomac.2022.12.251

Figure Lengend Snippet: PF 50 (MRJP2 with its isoform X1) neutralizes SARS-CoV-2 South African S protein (S Afr )-mediated pseudovirus entry and structures models. (a, c) Surface and cartoon representations of trimeric SARS CoV-2 S Afr (PDB ID: 7LYN , S1 “white”, RBD “brown”, S2 “turquoise”) with multivalent interactions of three PF 50 molecules (orange and yellow colored cartoons), followed by the human ACE2 (PDB ID: 1R42 “purple”). The two views of the docking model (a, c) are rotated by 180°. (b, d) The interacting amino acid residues (black) between SARS-CoV-2 S Afr -RBD (only the matching residues with S Afr RBD-ACE2 complex's interface pocket were presented) and the two molecules of (c) MRJP2 X1 (yellow) and one molecule of (b) MRJP2 (orange). The yellow-highlighted residue is one of the residues that are responsible for the high virulence and transmissibility of the South African SARS-CoV-2. The docked complex structure (a) was given by the PatchDock server ( https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php ) and was visualized by Discovery Studio 2020 Client software. (e) The binding affinity (ΔG) and other PDBePISA ( https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver ) results (hydrogen bonds “HB”, area, and residues number “ i N res ”) for the interfaces of the docked complexes obtained. (f) The heatmap clustered the number of RBD residues of S UK and S Afr that were bound to one, two, or three molecules of MRJP or three molecules of PF 50 . The pseudotyped SARS-CoV-2 S Afr was preincubated for 1 h with a three-fold serially diluted (g) PF 50 or (h) S1 neutralizing antibody (clone No. C-A11), and then the S Afr /PF 50 mixture was incubated for 48 h with ACE2-HEK 293 T cells. A luciferase assay system was utilized to evaluate the neutralization potencies of tested proteins. The half-maximal inhibitory concentration (IC 50 ) values were determined using GraphPad Prism version 9.2.0 by fitting the luciferase activity from serially diluted PF 50 or antibody to a sigmoidal dose-response curve. The cytotoxicity (red line) of PF 50 on the ACE2-HEK 293 T cells (g) was done using the cell titer-Glo luminescent cell viability assay. Data ( n = 3) is represented as the mean ± S.E.

Article Snippet: In addition, Thaw Medium 1 (#60187, growth medium without puromycin), anti-S antibody (#101024; clone No. C-A11), luciferase (Luc) reagent (#60690), and SARS-CoV-2 RdRp homogeneous assay kit (#78109) were supplied from BPS Bioscience.

Techniques: Software, Binding Assay, Incubation, Luciferase, Neutralization, Concentration Assay, Activity Assay, Cell Viability Assay

Journal: International Journal of Biological Macromolecules

Article Title: Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

doi: 10.1016/j.ijbiomac.2022.12.251

Figure Lengend Snippet: PF 50 (MRJP2 with its isoform X1) potently inhibits the activity of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) in vitro and structures models. (a, b) Dose-dependent inhibition of RdRp activity by PF 50 compared to 6-Chloropurine-ribose-5′-triphosphate (CPR—P), respectively. PF 50 was incubated with the purified RdRp (67 ng/μl) for 1 h, then the Alpha counts (the signal-to-noise ratio) that reflect the generation of the digoxigenin-labeled RNA duplex were measured. The half-maximal inhibitory concentration (IC 50 ) values were computed using GraphPad Prism version 9.2.0 by fitting the Alpha-screen intensity data from serially diluted PF 50 or CPR-P to a sigmoidal dose-response curve. Results are the mean ± S.E. of two independent experiments. (c) The complex structure of RdRp (green) and the NSP7 “deep blue”-NSP8 “light blue” dimeric protein (PDB ID: 7BV2 ). (d) Docking model of RdRp (PDB ID: 7BV2 ) with PF 50 (orange and yellow colored cartoons), followed by NSP7-NSP8 dimeric protein. The interacting residues in the docked complex are magnified and elucidate the binding of MRJP2 to the NSP8-binding sites on the RdRp (red residues). The docked complex of RdRp with PF 50 was given by the GRAMM-X ProteinProtein Docking Web Server ( http://vakser.compbio.ku.edu/resources/gramm/grammx/ ), while the docked complexes with NSP7-NSP8 dimeric complex were obtained by the PatchDock server ( https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php ). All the docked complexes were visualized by Discovery Studio 2020 Client software. (e) The binding affinity (ΔG) and other PDBePISA ( https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver ) results (hydrogen bonds “HB”, area, and residues number “ i N res ”) for the interfaces of the docked complexes obtained.

Article Snippet: In addition, Thaw Medium 1 (#60187, growth medium without puromycin), anti-S antibody (#101024; clone No. C-A11), luciferase (Luc) reagent (#60690), and SARS-CoV-2 RdRp homogeneous assay kit (#78109) were supplied from BPS Bioscience.

Techniques: Activity Assay, In Vitro, Inhibition, Incubation, Purification, Labeling, Concentration Assay, Binding Assay, Software

Journal: Frontiers in Microbiology

Article Title: H11/HSPB8 Restricts HIV-2 Vpx to Restore the Anti-Viral Activity of SAMHD1

doi: 10.3389/fmicb.2016.00883

Figure Lengend Snippet: AlphaScreen-based screening of virus–host proteins interactions. (A) Schematic representation of the overall screening procedure. (B) Schematic representation of the AlphaScreen-based luminescent system used to screen for human protein kinases that interact with HIV-2 Vpx. All recombinant proteins were produced in the wheat germ CFPS system. Briefly, recombinant FLAG-tagged HIV-2 Vpx or dihydrofolate reductase (DHFR) was incubated with each protein kinase. Protein A–conjugated acceptor beads with anti-FLAG antibody and streptavidin-coated donor beads were added and bound to the tagged substrate. Upon laser excitation, the donor beads convert ambient oxygen to singlet oxygen. When a molecular interaction occurs between HIV-2 Vpx and a particular kinase, singlet oxygen transfers to the acceptor beads, activating emission of light at 520–620 nm. (C) HIV-2 Vpx–interacting kinases identified from a human protein kinase library by AlphaScreen. The binding efficiency of HIV-2 Vpx with each kinase was normalized relative to the luminescence of control DHFR.

Article Snippet: Using the AlphaScreen IgG (protein A) detection kit (PerkinElmer), 15 μl of detection mixture containing 100 mM Tris-HCl [pH 8.0], 0.01% Tween-20, 1 mg/ml BSA, 5 μg/ml anti-FLAG antibody (GE Healthcare, Buckinghamshire, UK), 5 ng streptavidin-coated donor beads, and 5 ng anti-IgG (protein A) acceptor beads were added to each well, followed by incubation at 26°C for 1 h. AlphaScreen signals were detected on an EnVision device (PerkinElmer) using the AlphaScreen signal detection program.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Recombinant, Produced, Incubation, Binding Assay