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| 1. |
Heikkinen, L.S., Kazlauskas, A., Melén, K., Wagner, R., Ziegler, T., Julkunen, I. and Saksela, K.
(2008)
Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling.
J. Biol. Chem.
283
,
5719–27
.
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Notes:
The authors identified the N-terminal Src homology 3 (SH3) domain-binding motif of Crk and CrkL as the preferred binding partner of nonstructural protein 1 (NS1), an important virulence factor of the influenza A virus. Interaction of NS1 with Crk, CrkL and other SH3 domain-containing proteins p85, p85β and Eps8L1 was investigated by protein pull-down assays. Expression constructs for biotinylated Crk, CrkL, p85, p85β and Eps8L1 were created by amplifying the 123 amino acid biotin acceptor domain from Propionibacterium shermanii from the PinPoint Xa-T Vector and inserting it upstream of the protein-coding sequences in a pGEX vector derivative. These constructs and a construct encoding Myc-tagged NS1 were transfecting into 293FT cells, and the biotinylated proteins were immobilized from the cell lysate using TetraLink™ Tetrameric Avidin Resin. Any Myc-NS1 that bound to the immobilized protein was detected using Western Blot analysis and an anti-Myc antibody. The authors also investigated the ability of wildtype NS1 or NS1 mutants to inhibit interferon-induced gene expression. A reporter plasmid was created by cloning an interferon-stimulated response element upstream of a minimal thymidine kinase promoter driving firefly luciferase expression. A vector containing Renilla luciferase was used as a control to normalize transfection efficiency. Huh-7 cells were cotransfected with the firefly and Renilla luciferase reporter constructs (0.2µg and 5ng, respectively), treated with interferon-β and lysed using the Passive Lysis Buffer. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System.
(0003803) |
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Products: Dual-Luciferase® Reporter Assay System | TetraLink™ Tetrameric Avidin Resin |
| 2. |
Yuan, C., Sato, M., Lanier, S.M. and Smrcka, A.V.
(2007)
Signaling by a non-dissociated complex of G Protein betagamma and alpha subunits stimulated by a receptor-independent activator of G protein signaling, AGS8.
J. Biol. Chem.
282
,
19938–47
.
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Notes:
Activators of G protein signaling (AGS) proteins can activate G protein signaling in a G protein-coupled receptor-independent manner. To investigate the mechanism of this activation, the authors examined the ability of AGS8 to form a quaternary complex with Gα and Gβγ subunits and phospholipase (PLC) C β. Biotinylated Gβγ subunit was expressed and purified, then incubated with purified GST-ASG8, PLC Cβ and Gα. The complex was isolated using the TetraLink™ Tetrameric Avidin Resin, then subjected to Western blot analysis.
(0003779) |
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Products: TetraLink™ Tetrameric Avidin Resin |
| 3. |
Lovrich, S.D., La Fleur, R.L., Jobe, D.A., Johnson, J.C., Asp, K.E., Schell, R.F. and Callister, S.M.
(2007)
Borreliacidal OspC antibody response of canines with Lyme disease differs significantly from that of humans with Lyme disease.
Clin. Vaccine Immunol.
14
,
635–7
.
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Notes:
When infected with Borrelia burgdorferi, humans respond by producing borreliacidal antibodies against outer surface protein C (OspC). The authors examined the immune response in infected canines to determine if the response was similar. Infected dogs produced borreliacidal antibodies, only some of which were against OspC. To examine the borreliacidal activity of these other antibodies, sera from infected canines were depleted of OspC antibodies, and the borreliacidal activities of the depleted sera were measured. Sera were depleted by passage over an OspC column, which was created by immobilizing 0.5mg of biotinylated OspC protein with 1ml of TetraLink™ Tetrameric Avidin Resin.
(0003780) |
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Products: TetraLink™ Tetrameric Avidin Resin |
| 4. |
Igarashi, M., Yogiashi, Y., Mihara, M., Takada, I., Kitagawa, H. and Kato, S.
(2007)
Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene.
Mol. Cell. Biol.
27
,
7947-7954
.
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Notes:
Igarashi et al. showed that expression of the Msx2 gene, which enodes a transcription factor, is induced by vitamin K treatment via a pregnane X receptor response element (PXRE) and by estrogen via an estrogen response element (ERE). Promoter analysis was performed by cloning the Msx2 promoters into the pGL3-Basic Vector, transfecting MC3T3 and ST2 cells with the pGL3-Basic constructs, treating the cell with 10nM 17β-estradiol and 10µM vitamin K, then measuring luciferase activity. The pRL-CMV Vector (2.5ng per well of a 12-well plate) was cotransfected to normalize for transfection efficiency. The ability of PXR to bind to the Msx2-PXRE was assessed by an avidin-biotin complex DNA assay. Sense and antisense oligonucleotides that were biotinylated at the 5´ end were annealed and immobilized with the TetraLink™ Tetrameric Avidin Resin. HEK293T cells were lysed with lysis buffer (10mM Tris-HCl [pH7.8], 1mM EDTA, 150mM NaCl, 0.1% NP-40) containing protease inhibitors, then centrifuged to clarify the extract. The supernatants were mixed with the DNA-TetraLink™ Resin to allow proteins to bind to the oligos, and resin was washed with lysis buffer. Bound proteins were analyzed by SDS-PAGE and Western blotting.
(0003805) |
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Products: pGL3-Basic Vector | pRL-CMV Vector | TetraLink™ Tetrameric Avidin Resin |
| 5. |
Motohashi, H., Katsuoka, F., Miyoshi, C., Uchimura, Y., Saitoh, H., Francastel, C., Engel, J.D., and Yamamoto, M.
(2006)
MafG sumoylation is required for active transcriptional repression.
Mol. Cell. Biol.
26
,
4652–63
.
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Notes:
The authors examined the role of sumoylation in transcriptional repression by the homodimeric MafG protein and in transcriptional activation by a MafG/p45 heterodimer. To monitor transcription levels in the presence of MafG and mutated forms of MafG, the authors cotransfected 293T cells with a plasmid containing three copies of a Maf recognition site (MARE) driving expression of the firefly luciferase reporter gene, and an unspecified plasmid with the Renilla luciferase reporter gene for normalization. Reporter gene activities were measured using the Dual-Luciferase® Reporter Assay System. To examine DNA binding ability, polyhistidine-tagged MafG and MafG mutants were expressed in the TNT® Coupled Wheat Germ Extract System and used in electrophoretic mobility shift assays. The DNA-binding ability was also examined using biotinylated double-stranded DNA probes with a MARE sequence. The MafG proteins were allowed to bind to this probe, and the protein-DNA complexes were captured using the TetraLink™ Tetrameric Avidin Resin then separated by SDS-PAGE.
(0003660) |
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Products: Dual-Luciferase® Reporter Assay System | TNT® T7 Coupled Wheat Germ Extract System | TetraLink™ Tetrameric Avidin Resin |
| 6. |
Lovrich, S.D., Jobe, D.A., Schell, R.F., and Callister, S.M.
(2005)
Borreliacidal OspC antibodies specific for a highly conserved epitope are immunodominant in human lyme disease and do not occur in mice or hamsters.
Clin. Diagn. Lab. Immunol.
12
,
746–51
.
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Notes:
In early stage of Lyme disease in humans, the primary immune response is the production of antibodies to outer surface protein C (OspC). In previous work, the authors demonstrate that the antibody response is specific to the 50 amino acids nearest to the OspC C-terminus. In this study, the authors used smaller OspC fragments of 7 and 16 amino acids to more specifically locate the epitope. Proteins consisting of the final 7 C-terminal amino acids (C7) or 16 C-terminal amino acids (C16) were expressed as biotinylated proteins in E. coli. To purify C7 and C16, the E. coli cultures were sonicated and passed over columns of SoftLink™ Soft Release Avidin Resin at a rate of 0.5ml/minute at 4°C. The captured proteins were analyzed by SDS-PAGE and Western blot analysis using anti-OspC antibodies to confirm protein purity. An affinity column for OspC antibodies was generated by passing biotinylated C7 or C16 over a column of TetraLink™ Tetrameric Avidin Resin, which irreversibly binds biotinylated proteins. Human sera were passed over this column, and the flowthrough fractions were tested in an ELISA to determine the extent to which the OspC antibodies were adsorbed to C7 or C16 fragments.
(0003663) |
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Products: SoftLink™ Soft Release Avidin Resin | TetraLink™ Tetrameric Avidin Resin |
| 7. |
Kanamori, T., Kanou, N., Atomi, H. and Imanaka, T.
(2004)
Enzymatic characterization of a prokaryotic urea carboxylase.
J. Bacteriol.
186
,
2532–2539
.
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Notes:
These authors characterized urea carboxylase (Uca) from Oleomonas sagaranensis. The presence of a motif known to be biotinylated in biotin carboxyl carrier proteins implies that Uca is also biotinylated. Recombinant Uca was expressed in E. coli in the presence of 0.2mM biotin, and biotinylated Uca was partially purified using the TetraLink Tetrameric Avidin Resin according to the supplied protocol. The percent of Uca that was biotinylated was determined by comparing the amount of Uca that bound to the column (biotinylated) and the amount in the flowthrough (unbiotinylated). In this case, 60% of Uca was biotinylated.
(0003564) |
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Products: TetraLink™ Tetrameric Avidin Resin |
| 8. |
Despres, C., DeLong, C., Glaze, S., Liu, E. and Fobert, P.R.
(2000)
The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors.
Plant Cell
12(2)
,
279-290
.
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Notes:
The Arabidopsis NPR1 protein is part of a systemic and inducible plant defense mechanism and may be the plant homolog to IκBα, the NF-κB regulator. In a yeast two-hybrid screen, it was found that NPR1 interacts with several TGA factors. To verify these interactions, NPR1 was expressed and biotinylated in E. coli XL1-Blue using the Pinpoint™ Xa-1 Vector. The biotinylated protein was affinity-purified from a sonicated culture using TetraLink™ Tetrameric Avidin Resin. The captured NPR1 was assayed for interaction with TGAs produced and radiolabled with 35S-methionine. It was found that NPR-1 binds to TGA2 strongly and to TGA4 weakly. The TGA4 interaction was negetive in the yeast two-hybrid system. A C150Y transition mutant of NPR1 did not bind TGA2.
(0002707) |
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Products: TNT® T7 Quick Coupled Transcription/Translation System | TetraLink™ Tetrameric Avidin Resin |
| 9. |
Nobile, V. , Russo, N. , Hu, G. f. , Riordan, J. F.
(1998)
Inhibition of human angiogenin by DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex.
Biochemistry
37
,
6857-6863
.
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Notes:
The authors used SELEX to identify specific sequence aptamers that bind to angiogenin and inhibit both its ribonucleolytic and angiogenic activities. Various Promega products were used, including Taq DNA polymerase and TetraLink™ Tetrameric Avidin Resin, to isolate sequences amplified with biotin-tagged antisense primers. For assaying the effect of the aptamers on the activity of angiogenin, human umbilical vein endothelial cells were grown on fibronectin coated dishes in human endothelial serum-free medium supplemented with 20ng/ml basic FGF. Angiogenin was added to these cells with or without prior pre-mixing with aptamers.
(0000625) |
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Products: rhFGF, Basic | TetraLink™ Tetrameric Avidin Resin |