Bac-to-Bac™ Baculovirus Expression System

Citas y referencias (66)

Gibco™

Bac-to-Bac™ Baculovirus Expression System

El sistema de expresión de baculovirus Bac-to-Bac permite una producción eficaz de baculovirus recombinantes para la prueba de expresión enMás información

Número de catálogo	Cantidad
10359016	1 kit

Número de catálogo 10359016

Precio (CLP)

1.752.477

Añadir al carro de la compra

Cantidad:

1 kit

Precio (CLP)

1.752.477

Añadir al carro de la compra

El sistema de expresión de baculovirus Bac-to-Bac permite una producción eficaz de baculovirus recombinantes para la prueba de expresión en células de insectos. El sistema se basa en la generación de baculovirus recombinantes mediante la transposición específica de sitio en E. coli en lugar de la recombinación homóloga en células de insecto. Características del sistema:

• Bácmido de expresión que ahorra tiempo: con el sistema Bac-to-Bac, el casete de expresión del vector pFastBac se recombina con el bacmid padre en las células competentes de E. coli DH10Bac para formar un bacilo de expresión. A continuación, el bácmido se transfecta en células de insectos para producir partículas recombinantes de baculovirus.

• Fácil detección de colonias: el bácmido parental en la E. coli DH10Bac contiene un segmento del gen lacZa. El gen lacZa se interrumpe tras la transposición del casete de expresión en el bacilo, lo que permite la selección azul/blanca de recombinantes para facilitar la identificación de colonias recombinantes.

Alta eficacia de transfección con el reactivo de transfección ExpiFectamine SF: los kits de expresión TOPO Bac-to-Bac ahora incluyen la última generación de reactivos de transfección ExpiFectamine SF para una transfección de ADN eficaz en células de insectos utilizando protocolos rápidos y flexibles. Más información sobre este reactivo ›

• Flexibilidad: el sistema Bac-to-Bac incluye un vector pFastBac que contiene un sitio de clonación múltiple de gran tamaño (MCS) para la clonación simplificada. Se ofrecen otros dos vectores adicionales por separado.: Vector pFastBac HT para la producción de proteínas recombinantes marcadas con histidina y vector pFastBac Dual para la expresión de dos proteínas simultáneamente utilizando los promotores p10 y poliedrina.

• Alta expresión de proteínas: el vector pFastBac utiliza el potente promotor de poliedrina para generar altos niveles de expresión en una variedad de líneas celulares de insectos como Sf9, Sf21 y células High Five.

Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.

Especificaciones

Tipo de productoSistema de expresión de baculovirus

Cantidad1 kit

VectorpFastBac

Método de clonaciónEnzimas de restricción/MCS

Línea de productosBac-to-Bac

PromotorPoliedrina

Etiqueta de proteínaSin etiquetar

Unit SizeEach

Contenido y almacenamiento

El sistema de expresión en baculovirus Bac-to-Bac incluye:
• Vector pFastBac1 (10 μg), conservar a -20°C
• vector de control pFastBac 1-Gus (4 ng), conservar a -20°C
• E. coliquímicamente competente MAX Efficiency DH10Bac (1 kit con 5 x 0.1 ml), conservar a -80°C
• Reactivo de transfección ExpiFectamine Sf (1 ml), almacenar a 4 °C, no congelar

Preguntas frecuentes

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

Citations & References (66)

Citations & References

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