Bac-to-Bac™ Baculovirus Expression System

Zitierungen und Referenzen (66)

Gibco™

Bac-to-Bac™ Baculovirus Expression System

Katalognummer	Menge
10359016	1 Kit

Katalognummer 10359016

Preis (EUR)

1.738,00

Menge:

1 Kit

Preis (EUR)

1.738,00

Das Bac-to-Bac Baculovirus-Expressionssystem ermöglicht die effiziente Produktion von rekombinantem Baculovirus für Expressionstests in Insektenzellen. Das System beruht auf der Erzeugung des rekombinantem Baculovirus durch stellenspezifische Umsetzung in E. coli anstatt der homologen Rekombination in Insektenzellen. Dieses System hat folgende Merkmale:

• Zeitsparendes Expressions-Bacmid—Beim Bac-to-Bac-System rekombiniert die Expressionskassette des pFastBac-Vektors mit dem Parental-Bacmid in DH10Bac E. coli Kompetenten Zellen zu einem Expressions-Bacmid. Das Bacmid wird dann in Insektenzellen zur Produktion rekombinanter Baculovirus-Partikel transfiziert.

• Einfaches Kolonie-Screening—Das Parental-Bacmid in DH10Bac E. coli enthält ein Segment des lacZa-Gens. Das lacZa-Gen wird bei der Umsetzung der Expressionskassette in das Bacmid gestört, wodurch eine blau/weiße Selektion von Rekombinanten zur leichteren Identifizierung rekombinanter Kolonien möglich wird.

• Hohe Transfektionseffizienz mit ExpiFectamine Sf Transfektionsreagenz—Die Bac-to-Bac TOPO Expressionskits werden jetzt mit dem ExpiFectamine Sf Transfektionsreagenz der nächsten Generation für eine effiziente DNA-Transfektion in Insektenzellen mit schnellen, flexiblen Protokollen geliefert. Erfahren Sie mehr über dieses Reagenz ›

• Flexibilität—Das Bac-to-Bac System verfügt über einen pFastBac Vektor, der eine große MCS-Stelle (Multiple Cloning Site) für vereinfachtes Klonen enthält. Zwei weitere Vektorformate sind separat erhältlich: PFastBac HT-Vektor für die Produktion von Histidin-markierten rekombinanten Proteinen und pFastBac Dual-Vektor für die Expression von zwei Proteinen gleichzeitig unter Verwendung der p10- und Polyedrin-Promotoren.

• Hohe Proteinexpression—Der pFastBac Vektor verwendet den starken Polyhedrin-Promotor, um hohe Expressionsniveaus in einer Vielzahl von Insektenzelllinien wie Sf9, Sf21 und High Five Zellen zu erzeugen.

Nur für Forschungszwecke. Darf nicht für diagnostische Verfahren eingesetzt werden.

Specifications

ProdukttypBaculovirus-Expressionssystem

Menge1 Kit

VektorpFastBac

KlonierungsmethodeRestriktionsenzym/MCS

ProduktlinieBac-to-Bac

PromoterPolyhedrin

ProteinmarkierungNicht markiert

Unit SizeEach

Inhalt und Lagerung

Jedes Bac-to-Bac Baculovirus-Expressionssystem beinhaltet:
• PFastBac1 Vektor (10 µg), Lagerung bei -20 °C
• pFastBac 1-Gus Kontrollvektor (4 ng), Lagerung bei -20 °C
• MAX Efficiency DH10Bac Chemisch kompetente E. coli (1 Kit mit 5 x 0,1 ml), Lagerung bei -80 °C
• ExpiFectamine Sf Transfektionsreagenz (1 ml) Lagerung bei 4 °C, nicht einfrieren

Häufig gestellte Fragen (FAQ)

I cannot grow this white colony in liquid culture. What should I do?

The concentration of gentamicin might be too high. Try lowering the amount to 5 µg/mL and try adding more of the colony to the culture medium.

What has happened when I see blue colonies? How about colonies which are blue in the center and white on the edges?

In the case of a blue colony, the E. coli has the bacmid and the plasmid in it, allowing the cells to survive the selection process. However, because the transposition has not occurred, the LacZ gene is not disrupted. For bulls-eye colonies, this indicates that the transposition took place when the colony was growing. Re-streaking for an isolated clone from the white portion of the mixed colony should yield some colonies where transposition occurred.

I'm getting mostly white/wild-type plaques instead of blue/recombinant plaques. What am I doing wrong?

This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.

I've infected my cells and see large polyhedra in one cell and smaller polyhedra (more numerous) in a neighboring cell. Is this normal?

Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3-4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.

I'm worried that I am not getting plaques. How many days does it take to see plaques and what size are they typically?

Normally, very small white dots show up about 5-7 days and 1 mm plaques show up around day 10. Plaques can vary in size from 1 mm to 4 mm.

View all Bac-to-Bac™ Baculovirus Expression System FAQs

Zitierungen und Referenzen (66)

Zitierungen und Referenzen

Abstract

The vomeronasal receptor V2R2 does not require escort molecules for expression in heterologous systems.

Authors:Silvotti L, Giannini G, Tirindelli R,

Journal:Chem Senses

PubMed ID:15647459

'In rodents, many behavioural responses are triggered by pheromones. These molecules are believed to bind and activate two families of G-protein coupled receptors, namely V1Rs and V2Rs, which are specifically expressed in the chemosensory neurons of the vomeronasal organ. V2Rs are homologous with Group 3 of G-protein-coupled receptors, which includes ... More

Identification of Novel SH3 Domain Ligands for the Src Family Kinase Hck. WISKOTT-ALDRICH SYNDROME PROTEIN (WASP), WASP-INTERACTING PROTEIN (WIP), AND ELMO1.

Authors: Scott Margaret Porter; Zappacosta Francesca; Kim Eun Young; Annan Roland S; Miller W Todd;

Journal:J Biol Chem

PubMed ID:12029088

'The importance of the SH3 domain of Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, we used mass spectrometry to identify approximately 25 potential binding partners for the SH3 domain ... More

Allocation of helper T-cell epitope immunodominance according to three-dimensional structure in the human immunodeficiency virus type I envelope glycoprotein gp120.

Authors: Dai G; Steede N K; Landry S J;

Journal:J Biol Chem

PubMed ID:11551929

'The specificity and intensity of CD4(+) helper T-cell responses determine the effectiveness of immune effector functions. Promiscuously immunodominant helper T-cell epitopes in the human immunodeficiency virus (HIV) envelope glycoprotein gp120 could be important in the development of broadly protective immunity, but the underlying mechanisms of immunodominance and promiscuity remain poorly ... More

Expression, purification, and characterization of an activated cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase from baculovirus-infected insect cells.

Authors: Cai X Y; Shanahan M; Miller K; Gommoll C; Lundell D; Zavodny P; Dalie B;

Journal:Protein Expr Purif

PubMed ID:9226723

'An activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase was expressed in Spodoptera frugiperda (SF9) cells from a baculovirus vector. To maximize expression and to facilitate purification of the recombinant protein, CSBP2 kinase was expressed as a carboxy-terminal fusion protein to glutathione S-transferase (GST). Under optimal ... More

Kinesin Superfamily Motor Protein KIF17 and mLin-10 in NMDA Receptor-Containing Vesicle Transport

Authors:Mitsutoshi Setou, Terunaga Nakagawa, Dae-Hyun Seog, Nobutaka Hirokawa *

Journal:Science

PubMed ID:10846156

'Experiments with vesicles containing N-methyl-D-aspartate (NMDA)receptor 2B (NR2B subunit) show that they are transported alongmicrotubules by KIF17, a neuron-specific molecular motor in neuronaldendrites. Selective transport is accomplished by direct interaction of theKIF17 tail with a PDZ domain of mLin-10 (Mint1/X11), which is aconstituent of a large protein complex including mLin-2 ... More

View all Bac-to-Bac™ Baculovirus Expression System citations