We believe this site might serve you best:

United States

United States

Language: English

Promega's Cookie Policy

Our website uses functional cookies that do not collect any personal information or track your browsing activity. When you select your country, you agree that we can place these functional cookies on your device.

Our website does not fully support your browser.

We've detected that you are using an older version of Internet Explorer. Your commerce experience may be limited. Please update your browser to Internet Explorer 11 or above.

Citations Search

Sort By:

ACS Med. Chem. Lett. 5, 1190–1195. 1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain. 2014

Demont, E.H., Bamborough,P., Chung,C., Craggs, P.D., Fallon, D., Gordon, L.J., Grandi, P., Hobbs, C.I., Hussain, J., Jones, E.J., Le  A., Michon, A., Mitchell, D.J., Prinjha, R.K., Roberts, A.D., Sheppard, R.J, and Watson, R.J.

Notes: In this paper the authors report on the discovery, binding mode, and structure:activity relationship of the first potent, selective series of inhibitors of the BRPF1 (bromodomain and PHD finger-containing)  bromodomain.  Bromodomains are specific protein modules present in a group of chromatin-regulator proteins responsible for “reading” acetylated lysine residues. Although some bromodomain-containing proteins (BCPs), such as those in the BET subfamily, are well characterized and have been identified as potential therapeutic targets, other BCPs, including those in the BPRF subfamily, are less well understood.  These authors set out to generate selective inhibitors of the BRPF1 domain in order to better understand the functional role of this specific bromodomain region. Using an inhibitor discovery strategy based on other known compound-bromodomain interactions, a potent, selective inhibitor of the BRPF1 bromodomain was identified, synthesized, and characterized using in vitro methods.  To demonstrate the function of this compound in live cells, the NanoBRET™ assay for protein:protein interactions (PPI) was used. The NanoBRET™ PPI assay enabled the authors to demonstrate both the cell permeability of the newly identified compound and also the ability of the compound to disrupt chromatin binding of the BRPF1 domain. NanoLuc® Luciferase-tagged BRPF1 bromodomain and HaloTag®-labeled Histone H3.3 were used for the NanoBRET™ assay in HEK293 cells. Dose-response curves performed with the NanoBRET™ assay enabled calculation of the cellular IC50 of the newly identified compound. A less active control analog compound was unable to inhibit the BRPF1 bromodomain:Histone H3.3 interaction, demonstrating assay specificity. Finally, the newly identified compound was inactive in NanoBRET™ assays using a second BRPF1 isoform containing a natural insertion, a result that was consistent with the proposed compound mode of action. Confirmation that the new identified compound can enter cells and disrupt the BRPF1 bromodomain:chromatin interaction in a cellular environment suggests that it may be a useful compound for studying the physiological role and therapeutic potential of BCPs containing the BRPF1 bromodomain. (4514)

Expand Full Notes »

Proc. Natl. Acad. Sci. USA 112, 148–153. Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling. 2014

Wang, J., Ren, J., Wu, B., Feng, S., Cai, G., Tuluc, F., Peränen, J. and Guo, W.

Notes: To investigate whether protein conformation of Rabin8 plays a role in autoinhibition, the authors created a Rabin8 fusion construct with NanoLuc® luciferase at the N terminus and HaloTag® protein at the C terminus so that they can use BRET as an indication of protein conformation. A t-SRARE protein, syntaxin-4 (STX4), which is known to have a closed conformation, was constructed with the same NanoLuc® luciferase-STX4-HaloTag® protein configuration for use as a positive control. Both the control STX4 protein and Rabin8 were expressed in E. coli, the NanoBRET™ Nano-Glo® Substrate added and fluorescence measured. As a negative control, TEV protease was used to cleave the HaloTag® sequence from the protein fusions, eliminating the NanoBRET™ signal. NanoBRET™ signals were determined from experiments comparing Rabin8 with a gain-of-function Rabin8 mutant, exposing Rabin8 to constitutively active ERK2 or a kinase-dead ERK2 and assessing wildtype Rabin8 versus Rabin8-4D, where the aspartates acted as phosphorylation mimics. (4566)

Expand Full Notes »

J. Cell Sci. 127, 5261-72. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. 2014

Nakamura, T., Yoshitomi, Y., Sakai, K., Patel, V., Fukumoto, S. and Yamada, Y.

Notes: Epiprofin (Epfn) was expressed in HaCaT cells from various HaloTag® Fusion vectors with either full-length or deletion mutants of the CMV promoter to provide different levels of Epfn expression. (The authors do not state whether the N-terminal or C-terminal HaloTag® Fusion vectors were used). HaCaT 72hr proliferation was highest when using the CMVd3 promoter (e.g., pFC17A or pFN24A) for the lowest expression level. Reporter assays were performed on in the HaCaT cells as well and were measured with the Dual-Luciferase® Reporter Assay System on a GloMax® Instrument. Transfection studies performed with primary keratinocytes utilized the ViaFect™ Transfection Reagent using the reverse transfection method. (4690)

Expand Full Notes »

Proc. Natl. Acad. Sci. USA 111(38), 13990–5. Tyrosine phosphorylation of GluK2 up-regulates kainate receptor-mediated responses and downstream signaling after brain ischemia 2014

Zhu, Q.J., Kong, F.S., Xu, H., Wang, Y., Du, C.P., Sun, C.C., Liu, Y., Li, T. and Hou, X.Y.

Notes: In this study the authors looked for molecular mechanisms underlying the role of kainite receptors in ischemic stroke. In their studies, the researchers examined binding of Src kinase to GluK2, and the site of this interaction. A GST pulldown assay confirmed a direct interaction between GluK2 and Src in vitro. Then a bioluminescence resonance energy transfer (BRET) assay was used to examine this GluK2-Src interaction in live HEK293 cells, using NanoLuc® Luciferase as the energy donor, and HaloTag-labeled GluK2 as the energy acceptor. As reported, the co-expression of NLuc and HaloTag® fusions resulted in a significant NanoBRET ratio. The authors added untagged GluK2 as a competitor of the GluK2-HaloTag and Src-NLuc interaction, which resulted in reduction of the NanoBRET ratio. These results demonstrated that GluK2 interacts directly with Src in living cells. The GloMax® Discover Detection System was used to measure the output of these assays.

Their source of NanoLuc® Luciferase and HaloTag® tags was the NanoBRET™ PPI Starter System (Cat.# N1811, N1821). (4696)

Expand Full Notes »

Protein Expr. Purif. 89, 62–72. Application of HaloTag technology to expression and purification of cannabinoid receptor CB2. 2013

Locatelli-Hoops, S., Sheen, Fangmin C., Zoubak, L., Gawrisch, K. and Yeliseev, A.A.

Notes: The HaloTag® Protein Tag was used to tag, immobilize and purify functional cannabinoid receptor CB2, a GPCR,  after induction of expression in E. coli.  (4263)

Expand Full Notes »

Cell 153, 1327-1339. HIF1A Employs CDK8-Mediator to Stimulate RNAPII Elongation in Response to Hypoxia. 2013

Galbraith, M., Allen, M., Bensard, C., Wang, X., Schwinn, M., Qin, B., Long, H., Daniels, D., Hahn, W., Dowell, R., and Espinosa, J.

Notes: These authors identified a previously unknown interaction between the transcription factor HIF1A and the cyclin-dependent kinase CDK8 (a component of the Mediator complex) in the regulation of genes associated with cellular survival under low-oxygen conditions. As part of the study, HaloTag technology was used to identify proteins interacting with CDK8 in a colorectal cancer cell line. Specifically, cells were transfected with CDK8 and CDK19 HaloTag fusion constructs obtained from Kazusa Institute. The cell lysates were then used in pulldown assays to capture interacting proteins. The results showed that CDK8 and CDK19 are present in mutually exclusive Mediator complexes. Details of the transfection are as follows: HCT116 cells were plated in 150 mm dishes and grown to 70%–80% confluence before transfection with 30 μg of plasmid DNA using FuGENE HD Transfection Reagent. (4355)

Expand Full Notes »

Cell 154, 541–555. KDM4A Lysine Demethylase Induces Site-Specific Copy Gain and Rereplication of Regions Amplified in Tumors 2013

Black, J.C., Manning, A.L., Van Rechem, C., Kim, J., Ladd, B., Cho, J., Pineda, C.M., Murphy, N., Daniels, D.L., Montagna, C., Lewis, P.W., Glass, K., Allis, C.D., Dyson, N.J., Getz, G. and Whetstine, J.R.

Notes: The HaloTag® protein tag was used in experiments to identify protein partners of KDM4A that involved in site-specific copy number gain in tumors, specifically at the 1q12h region. Expression constructs were transfected into HEK293T cells using the FuGENE® HD Transfection Reagent. The HaloTag-KDM4A (Cat.# FHC00602) and HaloTag-Suv39h1 (Cat.# FHC09879) expression constructs were obtained from the Kazusa DNA Research Institute (Kisarazu, Japan). Interacting proteins identified included DNA polymerase subunits and members of the minichromosome maintenance (MCM) complex. (4408)

Expand Full Notes »

EMBO J. 32, 645–55. TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS. 2013

Deplus, R., Delatte, B., Schwinn, M.K., Defrance, M., Méndez, J., Murphy, N., Dawson, M.A., Volkmar, M., Putmans, P., Calonne, E., Shih, A.H., Levine, R.L., Bernard, O., Mercher, T., Solary, E., Urh, M. Daniels, D. and Fuks, F.

Notes: These authors set out to determine how TET2 and TET3 proteins are involved in epigenetic regulation. To characterize proteins that interact with TET, the authors expressed full-length TET1, TET2 and TET3 as HaloTag® fusion proteins and performed protein pull-downs. They identified novel interactions between all three TET proteins and O-GlcNAc transferase (OGT), which catalyzes the addition of N-acetylglucosamine (GlcNAc) to numerous transcription factors, regulatory proteins and histones to activate or inhibit the target protein or recruit additional proteins. In this paper, they focused on TET2 and TET3, which showed the strongest interaction with OGT. They mapped TET2, TET3 and OGT binding throughout  the genome by expressing these proteins as HaloTag® fusion proteins in HEK293T cells, crosslinking the proteins and DNA, then capturing the fusion proteins and associated DNA fragments and performing high-throughput sequencing to show that TET2/3 and OGT colocalize at active gene promoters and were tightly clustered near transcription start sites.

For expression of HaloTag® fusion proteins and controls, HEK-293 cells were plated at 12 ×106 cells in a 150mm dish and grown to 70–80% confluency before transfection with 30µg of plasmid using the FuGENE® HD Transfection Reagent.

To assess whether TET2/3-OGT activity affects the interaction of SET1/COMPASS with chromatin, the authors used bioluminescence resonance energy transfer (BRET). They created a fusion protein consisting of the H3K4 methyltransferase SETD1A and NanoLuc® luciferase as the energy donor and a fluorescently labeled histone H3.3-HaloTag® fusion protein as the energy acceptor.  These BRET experiments confirmed that TET2/3-OGT activity is necessary for SET1/COMPASS complex function and showed that TET and OGT activities promote binding of SETD1A, a component of the SET1/COMPASS complex, to chromatin. This binding increases H3K4me3 levels. Thus, the authors’ data support a TET2/3-OGT-mediated mechanism for regulating the SET1/COMPASS complex and thus H3K4me3. (4262)

Expand Full Notes »

Current Chemical Genomics 6, 72-78. HaloTag, a Platform Technology for Protein Analysis. 2012

Urh, M., and Rosenberg, M.

Notes: This paper provides an overview of the many applications of HaloTag® Technology. The authors describe the development of the technology, focusing on it's multifunctional utility for protein imaging, protein isolation and display, and in the study of protein complexes and interactions. They also discuss it's potential to facilitate proteomics research studies across complex biological systems at the biochemical, cell-based and whole animal level. (4325)

Expand Full Notes »

J. Bacteriol. 194, 1389-1400. Legionella pneumophila LidA affects nucleotide binding and activity of the host GTPase Rab1. 2012

Neunuebel, M.R., Mohammadi, S., Jarnik, M., and Machner, M.P.

Notes: In this study, the L. pneumophila protein Lem3 was expressed as a HaloTag® fusion protein and purified using HaloLink™ Resin. Lem3 was first cloned into the pFN22K HaloTag® Vector and the resultant HaloTag-Lem3 protein was expressed in Single-Step (KRX) competent cells before purification using the HaloTag® Protein Purification System. Lem3 was cleaved from the HaloLink™ Resin using TEV protease.


Expand Full Notes »

Sci. Signal. 4(180), online. Identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response 2011

Yuanzheng, H., Xu, Y., Zhang, C., Gao, X., Dykema, K.J., martin, K.R., Ke, J., Hudson, E.A., Khoo, S.K., Resau, J.H., Alberts, A.S., Mackeigan, J.P., Furge, K.A. and Xu, H.E.

Notes: Yuangheng He and colleagues asked how the weak alkaline compound chloroquine (CQ) enhances the anti-inflammatory effects of synthetic glucocorticoids like dexamethasone, which are used to treat a host of inflammatory and autoimmune diseases. In the process they explored the intersection of lysosomal degradation pathways and glucocorticoid receptor signaling. They used Dual-Glo® Luciferase Assay System to look at glucocorticoid receptor-mediated (GR) activation and repression of reporters in AD293 cells under a variety of conditions (presence or absence of CQ; stripped serum, loss of lysosome synthesis, inhibition of V-ATPase, etc). They also used HaloTag® protein fusions to observe the fate of GR populations in the presence or absence of CQ and in the presence or absence of compounds that impair proteasome function. Live-cell imaging of GR-HaloTag® protein fusions revealed a dynamic association of the GR with lysosomes. The authors showed that glucocorticoid signaling is regulated by lysosomes. (4203)

Expand Full Notes »

Nat Chem. Biol. July 3, Epub. Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. 2011

Neklesa, T.K., Tae, H.S., Schneekloth, A.R., Stulberg, M.J., Corson, T.W., Sundberg, T.B., Raina, K., Holley, S.A., and Crews, C.M.

Notes: These authors investigated whether HaloTag® technology could be used to specifically target and degrade intracellular proteins. They developed a method to degrade specific proteins of interest using a small molecule by tagging the HaloTag® protein with an adamantyl moiety. The hypothesis was that appending a hydrophobic residue to a protein surface domain would mimic partial denaturation and induce proteasomal degradation. They first used a HaloTag®-luciferase fusion protein to determine the biological activity of various small molecules that enhanced hydrophobicity. After selecting the small molecule that reduced luciferase activity in HEK293 cells, they went on to test their hypothesis in various in vitro and in vivo models. They were able to demonstrate degradation of various HaloTag®-linked transmembrane proteins expressed in HEK293 cells, to show degradations of target proteins in Zebrafish, and to show degradation of the HRAS oncogene product both in NIH3T3 cells and in a mouse tumor model. (4125)

Expand Full Notes »

Biochem. J. 436, 387–397. The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response. 2011

Maruyama, A., Nishikawa, K., Kawatani, Y., Mimura, J., Hosoya, T., Harada, N., Yamamato, M. and Itoh, K.

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag® CMV Flexi® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase® Reporter Assay System. (4123)

Expand Full Notes »

Protein Expr. Purif. 76, 154-164. HaloTag-based purification of functional human kinases from mammalian cells. 2010

Ohana, R.F., Hurst, R., Vidugiriene, J., Slater, M.R., Wood, K.V. and Urh, M.

Notes: The authors of this paper demonstrate the utility of the HaloTag® protein purification system for purifying functional proteins from mammalian cells. To this end five kinases were cloned into HaloTag® vectors, expressed in and purified from HEK293T cells. To demonstrate functionality of the purified recombinant kinases, activity was measured using the appropriate ADP-Glo™ Assay Kinase Enzyme Systems. (4145)

Expand Full Notes »

Development 137, 901–11. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. 2010

Hattori, T., Müller, C., Gebhard, S., Bauer, E., Pausch, F., Schlund, B., Bösl, M.R., Hess, A., Surmann-Schmitt, C., von der Mark, H., de Crombrugghe, B. and von der Mark, K.

Notes: To study the role of the transcription factor Sox9 in the transition from cartilage to bone in newborn mice, the authors performed chromatin immunoprecipitation using the HaloCHIP™ System. Primary rib chondrocytes were transfected with a vector expressing full-length Sox9 with a HaloTag® protein tag, then proteins and DNA were cross-linked. DNA was isolated and sonicated, and the Sox9:DNA complexes were precipitated using the HaloLink™ Resin. The precipitated DNA then was amplified by PCR to determine that SOX9 binds to the SRY sites in the Vegfa gene. (4054)

Expand Full Notes »

BMC Genomics 10, 497. A functional analysis of the CREB signaling pathway using 2009

Danette D Hartzell, Nathan D Trinklein, Jacqui Mendez, Nancy Murphy, Shelley F Aldred, Keith Wood and Marjeta Urh

Notes: The HaloCHIP System was used to measure CREB binding as an alternative to the ChIp method. (4057)

Expand Full Notes »

Chem. Commun. 27, 4040-42. Cell-based in vivo dual imaging probes using genetically expressed tags and chemical contrast agents. 2009

Hasegawa, Y., Honda, A., Umezawa, K., Niino, Y., Oka, K., Chiba, T., Aiso, S., Tanimoto, A., Citterio, D., and Suzuki, K.

Notes: These authors describe a method for cell-surface labeling with two genetically expressed tags, a biotin acceptor peptide (BAP) and HaloTag (HT). BAP and HT were fused with the N-terminus of the mouse Ig κ-chain leader sequence to direct them to the secretory pathway, and the c-terminus of the platelet-derived growth factor receptor transmembrane domain to anchor them to the plasma membrane. Fluorescent proteins were fused with BAP and HT to create the two distinct fluorescent transmembrance proteins BAP-YFP-TM and HT-CFP-TM. The constructs were introduced into HeLa cells and imaged simultaneously. The authors then demonstrated in vivo imaging by injecting the doubly-tagged HeLa cells into nude mice and imaging with near infrared fluorescent probes, and with magnetic resonance probes. (3998)

Expand Full Notes »

Protein Expr. Purif. 68(1), 110-120. HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification 2009

Ohana, R.F., Encel, L.P., Zhao, K., Simpson, D., Slater, M.R., Urh, M., Wood, K.V.

Notes: The authors note that while many protein fusion tags are available to assist in purifying functional, recombinant proteins in soluble form and adequate amounts, none of the available tags are ideal when applied to the diversity of proteins studied. Available tags are often best-suited to specific aspects of the overall process, be it expression, solubilization, protein capture, etc. HaloTag was designed to address multiple features. The authors demonstrated that HaloTag provided enhanced expression and solubility in E. coli, with efficient protein purification and labeling for screening and quantitation. Enhanced solubility coupled with covalent capture gives HaloTag advantages over conventional affinity fusion tags, demonstrated by the successful purification of a wide variety of proteins, including low expressers, with higher yield and purity compared to the other tested tags. Covalent capture coupled with efficient tag removal to release the target protein, provides higher purity by eliminating tag contaminants. Using full-length cDNAs encoding 23 human proteins of varying size and function, the authors compared the efficacy of HaloTag as an expression and purification tag to the frequently used solubility enhancers MBP and GST. As reported previously, the three larger tags (HaloTag, MBP and GST) provided higher total and soluble expression compared to the smaller His6tag. In comparing soluble protein expression levels for the three larger tags, it was found that HaloTag solubilized 74% of the 23 human proteins examined, compared to 52% for MBP and 39% for GST. G2681 was one of the T7, bacterial promoter-based Flexi vectors used.

Expand Full Notes »

Methods in Mol. Biol. 577, 25-39. High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins 2009

Yamakawa, Hisashi

Notes: The authors use the Flexi® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag® Technology. They also describe a method of transferring ORFs between Flexi® Vectors in a 96-well plate format. They also used Wizard® SV 96 Plasmid DNA Purification, Wizard® SV PCR Clean-Up, and Wizard® SV Gel and PCR Clean-Up Systems. (4056)

Expand Full Notes »

Biophys. J. 96, L01-L03. In vivo labeling method using genetic construct for nanoscale resolution microscopy. 2009

Schröder, J. Benink, H., Dyba, M. and Los, G.V.

Notes: Traditionally light microscopy resolution has been limited by the diffraction of light. However several new technologies have emerged that partially overcome that limitation. One of these stimulated emission depletion (STED) microscopy is now commercially available and has been integrated into confocal microscope platforms. Because STED depends on fluorescent markers that fulfill specific spectroscopic needs, its uses have been limited. The authors of this paper demonstrate successful high resolution of β1-integrin-Halotag®-fusion protein distribution using STED microscopy. Use of the HaloTag® technology allows researchers to create a reporter that can be labeled with STED-optimized fluorescent tags. (3955)

Expand Full Notes »

J. Clin. Endocrinol. Metab. 94, 2594-2601. KLF15 Is a transcriptional regulator of the human 17beta-hydroxysteroid dehydrogenase type 5 gene. A potential link between regulation of testosterone production and fat stores in women 2009

Du, X., Rosenfield, R. and Qin, K.

Notes: The authors used a HaloTag® vector and the HaloCHIP™ System to identify a KLF15 binding site in the HSD17B5 promoter. (4059)

Expand Full Notes »

J. Clin. Endocrinol. Metab. 94, 2650-2657. Posttranscriptional Regulation of CDC25A by BOLL Is a Conserved Fertility Mechanism Essential for Human Spermatogenesis 2009

Yung Ming Lin, Chia Ling Chung, and Yu Sheng Cheng

Notes: Human boule (BOLL) gene was subcloned into the HaloTag® pHT2 Vector and transformed into JM109 and HeLa cells. (4060)

Expand Full Notes »


Anal. Biochem. 392, 45-53. Protein-protein interaction studies on protein arrays: effect of detection strategies on signal-to-background ratios. 2009

Hurst, R., Hook, B., Slater, M.R., Hatrnett, J., Storts, D.R., and Nath, N.

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)35S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

Expand Full Notes »

Methods in Mol. Biol. 577, 121-131. Pulse-Chase Experiment for the Analysis of Protein Stability in Cultured Mammalian Cells by Covalent Fluorescent Labeling of Fusion Proteins 2009

Kei Yamaguchi, Shinichi Inoue, Osama Ohara and Takahiro Nagase

Notes: The authors used the Halotag® Labeling Technology in pulse-chase experiments. They pulse labeled proteins in cultured mammalian cells. Using the HaloTag® Technology, they were able to monitor the degradation of the labeled protein, Smad1, that was induced by coexpression of Smurf1. They conclude that the HaloTag® Technology could be used to monitor the regulation of SMAD1 degradation. (4055)

Expand Full Notes »

Proc. Natl. Acad. Sci. USA 106, 5669-5674. Regulation of the processivity and intracellular localization of Saccharomyces cerevisiae dynein by dynactin. 2009

Kardon, J.R., Reck-Peterson, S.L. and Vale, R.D.

Notes: These authors expressed recombinant dynactin and dynein in Saccharomyces cerevisiae and investigated their interactions in motility assays. They created a c-terminal Halotag-Dynactin fusion, and were able to site-specifically label the fusion protein with the fluorescent dye tetramethylrhodamine (TMR). They studied the effect of the purified dynactin fusion protein on the motility of dynein complexes using total internal reflection fluorescence microscopy. Dynactin alone did not interact with microtubules. However, when coincubated with recombinant dynein, the TMR-labeled dynactin moved processively along microtubules. The authors then used truncation mutants of dynactin to identify the region of the dynactin molecule required for localization and enhanced processivity of dynein. (3960)

Expand Full Notes »