Shop All Protein Expression Kits

Freedom™ DG44 Kit (Gibco™)

Gibco® Freedom® DG44 Kit is an easy-to-use, beginning-to-end product, for cloning and expression of recombinant proteins in dihydrofolate reductase deficient (DHFR-) Chinese Hamster Ovary cells. The Gibco® Freedom® DG44 Kit includes components for transfection, expression, clone creation, and stable cell line selection, all in a conveniently packaged kit. The Gibco® Freedom® DG44 Kit also delivers a proven host cell line with simplified commercial licensing options.

• Flexible commercial licensing without the need for royalties
• Proven host used in approved products and a wide array of proteins
• Complete workflow that can take your gene of interest from transfection to lead clone typically in less than 6 months
• Convenient packaging, for simplified ordering and storage

Commercial Licensing that is Simple, Flexible and Without Royalties
Multiple companies provide CHO (Chinese Hamster Ovary) stable cell line development services and products that require annual fees during development of your therapeutic protein. In addition, royalties may be required on the sales of your therapeutic protein upon commercialization. Gibco® Freedom® Kits provide you with the tools that enable you or your CRO provider to develop your stable cell line for just the price of the kit itself. Furthermore, as you move towards commercial use, a flexible license structure is available without the burden of royalties and works to keep it as simple as a onetime payment (Fig 1). For more information regarding licensing, email us at outlicensing@lifetech.com.

Host Cell Background with Proven Results
DG44 cells have been a choice of scientists for decades for the expression of IgG and numerous blood proteins. Protein expression levels can vary significantly depending on the class of protein, however gram per liter levels of IgG expression has been demonstrated using these DG44 parental cells. So you can be confident that if your work calls for use of DG44 cells, they will deliver expected protein levels.

Complete, Fast, and Efficient Workflow
The Gibco® Freedom® DG44 Kit’s complete, proven, and fully integrated workflow allows you to move from transfection to lead clone typically in less then 6 months, helping to reduce hands on time and Fulltime Equivalent (FTE) requirements (Fig 2). We have implemented a single round of MTX (methotrexate) amplification in the protocol, which in our hands provided up to 120 fold titer improvement in less than 6 weeks (Fig 3). The entire workflow is performed in animal origin free (AOF) medium. The kit also offers the following advantages:

• Cloning of one or two genes that encode your protein(s) of interest
• High efficiency transfection of DNA into DG44 Cells (cGMP-banked)
• Included flash drive with loaded manual

New Convenient Packaging
To free you to focus on the creation of your stable cell line rather than on figuring out where and when all the components of your experiment are arriving, Freedom® DG44 Kit introduces the smART™ tote packaging system (shown in above product photos). All components for the creation of your expression cell line are delivered in soft cooler totes along with a parts flyer, and a flash drive containing the manual. Upon delivery you have all the information needed for correct storage and use of components consolidated in the two smART™ tote shipment coolers.

Access to Gibco® Freedom® Kit Support
If any specific support is needed beyond the manual, please contact your account manager to obtain a Process Science Manager consultation. In addition, one of our support scientists may check in with you to ensure that your experience with the kit is as successful as possible. For further assistance, please email our support team at GibcoServices@thermofisher.com.

Bac-to-Bac™ HBM TOPO™ Cloning Kit (Gibco™)

Bac-to-Bac® Baculovirus Expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies on generation of recombinant virus by site-specific transposition in E. coli (DH10Bac™) rather than homologous recombination in insect cells. The new pFastBac™ HBM TOPO® vector enables secreted protein expression because it has the honeybee melittin (HBM) secretion signal. The new vector should be strongly considered for the expression of glycoproteins. Glycoproteins cannot be glycosylated in the absence of any secretion signal. In contrast to glycoproteins secreted from mammalian cells, glycoproteins secreted from baculovirus can be easily de-glycosylated in vitro. This is a very important feature in order to crystallize proteins!

The pFastBac™ HBM TOPO® vector has also a C-terminal His-Tag with a TEV cleavage signal to enable easy purification of Histidine fusion proteins on nickel-chelating resins (ProBond™ Purification System) and native proteins with the aid of AcTev™ protease.
The vector uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell lines such as Sf9, Mimic™ Sf9, Sf21, and High Five™ cells in Sf-900 II & Sf-900™ III media from Gibco.

Traditional baculovirus systems require purification and amplification of an initial low-titer viral supernatant. This requires a time-consuming plaque assay. Bac-to-Bac®'s pFastBac™ vectors, however, recombines with the parent bacmid in DH10Bac™ E. coli competent cells to form an expression bacmid. Transfect the bacmid into insect cells for fast production of a high titer of pure recombinant baculovirus particles in the very first transfection. You'll save weeks of precious time. Collect a pure P2 baculovirus stock on Day 10 without the necessity of tedious plaques assay.

Bac-to-Bac™ HBM TOPO™ Secreted Expression System (Gibco™)

Bac-to-Bac® Baculovirus Expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies on generation of recombinant virus by site-specific transposition in E. coli (DH10Bac™) rather than homologous recombination in insect cells. The new pFastBac™ HBM TOPO® vector enables secreted protein expression because it has the honeybee melittin (HBM) secretion signal. The new vector should be strongly considered for the expression of glycoproteins. Glycoproteins cannot be glycosylated in the absence of any secretion signal. In contrast to glycoproteins secreted from mammalian cells, glycoproteins secreted from baculovirus can be easily de-glycosylated in vitro. This is a very important feature in order to crystallize proteins!

The pFastBac™ HBM TOPO® vector has also a C-terminal His-Tag with a TEV cleavage signal to enable easy purification of Histidine fusion proteins on nickel-chelating resins (ProBond™ Purification System) and native proteins with the aid of AcTev™ protease.
The vector uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell lines such as Sf9, Mimic™ Sf9, Sf21, and High Five™ cells in Sf-900 II & Sf-900™ III media from Gibco.

Traditional baculovirus systems require purification and amplification of an initial low-titer viral supernatant. This requires a time-consuming plaque assay. Bac-to-Bac®'s pFastBac™ vectors, however, recombines with the parent bacmid in DH10Bac™ E. coli competent cells to form an expression bacmid. Transfect the bacmid into insect cells for fast production of a high titer of pure recombinant baculovirus particles in the very first transfection. You'll save weeks of precious time. Collect a pure P2 baculovirus stock on Day 10 without the necessity of tedious plaques assay.

1-Step Human Coupled IVT Kit - DNA (Thermo Scientific™)

Thermo Scientific 1-Step Human In Vitro Protein Expression Kits enable the translation and post-transcriptional modification of full-length proteins from mRNA or plasmid templates with yields of up to 100 µg/mL per reaction.

The Human IVT Kits are unique HeLa cell lysate-based protein expression systems for in vitro translation or coupled transcription/translation reactions. Protein expression is performed in a single 90-minute reaction that can be extended for up to 6 hours with continued protein production up to 100 µg/mL when combined with the optimized pT7CFE1 Expression Vector. The Human IVT Kits can express functional enzymes, phosphoproteins, glycoproteins and membrane proteins for immediate use in studying protein interactions, performing rapid mutational analysis and measuring activity.

Features of the 1-Step Human Coupled IVT Kit:

Functional—uses the human translational machinery to express active proteins
Convenient—perform transcription and translation in a single step
High performance—greater yields compared to rabbit reticulocyte in vitro translation
Reliable—express proteins that fail in rabbit reticulocyte systems

Better Than Traditional Methods:
• HeLa cell-free extracts are capable of expressing proteins with post-translational modifications
• Accurate translation delivers full-length protein compatible with downstream applications
• Protein translation is optimized with EMCV IRES element and other mRNA stabilizing elements

Includes:
• Complete kits contain HeLa cell lysate, accessory proteins, reaction mix, nuclease-free water, expression vector and a GFP-positive control vector

Requires:
• 30°C incubator or water bath

Applications:
• Express proteins to measure enzyme activity
• Perform mutational analysis
• Express protein for use in gel mobility shift assays
• Perform co-immunoprecipitation
• Express cytotoxic proteins
• Perform unnatural amino acid labeling

The 1-Step Coupled Human IVT Kit for DNA is a cell-free system using the cellular transcription and translation machinery from a modified HeLa cell line. The procedure is quick and easy and is an effective alternative to other protein expression methods. Simply add an appropriate expression construct to a mixture of HeLa cell lysate, Accessory Proteins, Reaction Mix and incubate at 30°C for 90 minutes to overnight.

The 1-Step Human IVT Kit for mRNA also uses the human protein translation machinery to produce functional protein. This kit is recommended for translation of mRNA transcripts containing an EMCV IRES element and mRNA stabilizing features designed into the pT7CFE1 expression vector.

The human in vitro translation system is robust and will express proteins from a variety of species including mammals, bacteria and protozoa. Benchmarking shows that the expression levels of functional proteins such as luciferase are much higher in the human system compared to either rabbit reticulocyte-based or E. coli-based systems. Furthermore, proteins expressed with the human in vitro translation system are not contaminated with substances that can interfere with downstream applications.

More Product Data
Protocol for in vitro protein expression using mRNA templates
Glycoprotein expression in a human IVT system

Jump In™ T-REx™ CHO-K1 Kit

The Jump-In™ T-REx™ CHO-K1 Kit allows the targeted integration of genetic material into a specific pre-engineered R4 site in the Jump-In™ CHO-K1 cell line, to create an inducible isogenic stable cell line with less effort and in less time than traditional cell engineering methods. These cells stably express the tetracycline repressor protein, allowing for inducible expression of your retargeted gene of interest upon the addition of doxycycline.

The high retargeting efficiency, made possible by the R4 sites in the CHO-K1 cell line, allows the use of the isogenic pool for additional experiments without the need for clonal selection. Alternatively, the high retargeting efficiency allows for the easy selection of a positive stable clone expressing your gene of interest.

The Jump-In™ T-REx™ CHO-K1 Kit lets you:

• Quickly and efficiently develop stably engineered isogenic cell pools in about half the time compared to traditional cell engineering methods
• Inducibly express your gene of interest using doxycycline
• Utilize isogenic expression from a defined genomic locus as the ideal solution for comparative analysis of gene families, isoforms, or orthologs
• Generate multiple cell lines in parallel using the simplified work flow
• Easily access the technology without complicated licenses or restrictions to interpret

Save Time with Rapid and Efficient Generation of Engineered Cell Lines
With the Jump-In™ T-REx™ CHO-K1 Kit you can generate functional cell pools in as little as 2 weeks without laborious clone isolation and analysis, and the streamlined workflow makes it easier to generate several cell lines at the same time. Even generation of clonal cell lines can be done with reduced time and effort due to the high percentage of positive clones. In addition, the Jump-In™ technology gives you the freedom to generate an unlimited number of cell lines without restrictive licensing requirements.

Expand Your Experimental Capabilities
The Jump-In™ T-REx™ CHO-K1 Kit allows for inducible expression of your toxic or unstable gene of interest and is the ideal solution for targets and assays where transient engineering technologies are problematic. The kit also provides a convenient way to create target panels of gene families, isoforms, or orthologs. Genes coding for large proteins or multi-unit proteins are not a problem since the Gateway® destination vectors accept large inserts.

The kit includes:

Jump-In™ T-REx™ CHO-K1 Cells (2 vials @ 1 ml each)
pJTI™ R4 Dest CMV TO pA Vector (100 µg)--A Gateway® Destination vector that drives inducible expression of your gene of interest under control of the strong CMV promoter by inclusion of the Tet-Operon.
pJTI™ R4 Int Vector (100 µg)--Expresses the R4 integrase to facilitate the retargeting of your gene of interest into the R4 site.

Sold separately:

pJTI™ R4 CMV-TO MCS pA Vector--A multi-site cloning vector that drives inducible expression of your gene of interest under control of the strong CMV promoter by inclusion of the Tet-Operon.
pJTI™ R4 EXP CMV-TO EmGFP pA Vector--A control vector with inducible expression of GFP under control of the strong CMV promoter by inclusion of the Tet-Operon.

MembranePro™ Functional Protein Expression Kit (Invitrogen™)

For customers studying GPCRs and other membrane proteins, MembranePro™ is the ready-to-use system that delivers enriched, functional membrane proteins efficiently and reliably. Benefits of MembranePro™ include:

• Proteins are displayed on human (or other mammalian) cell membranes
• Cellular quality control and mammalian posttranslational processing helps produce functional protein
• Proteins bud off as lipoparticles and are easily collected from the culture medium
• Lipoparticles are enriched with expressed receptors

MembranePro™ workflow is less labor intensive in comparison to traditional production of membrane fractions. Briefly, the MembranePro™ protocol allows you to:

1. Clone your gene of interest using a TOPO® vector
2. Transfect with Lipofectamine™ 2000 and MembranePro™ Reagent into 293FT cells
3. Harvest lipoparticles 48h post-transfection, concentrate with MembranePro™ Precipitation Mix
4. Store lipoparticles at -80 C or proceed to downstream assays

MembranePro™ products are offered in two configurations – as an expression kit (TOPO® vector, Lipofectamine™ 2000, MembranePro™ Precipitation Mix, MembranePro™ Reagent, 293FT cells) and as a support kit (Lipofectamine™ 2000, MembranePro™ Precipitation Mix, MembranePro™ Reagent). The expression kit is only available in a 10 reaction size; the support kit is available in 10, 60 and 600 reaction sizes.

Bac-to-Bac™ C-His TOPO™ Cloning Kit (Gibco™)

Bac-to-Bac® Baculovirus Expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies on generation of recombinant virus by site-specific transposition in E. coli rather than homologous recombination in insect cells. New: pFastBacTOPO® vectors!


Reliable and fast protein expression
The Bac-to-Bac® Baculovirus Expression System is faster and easier than traditional baculovirus expression, and it maintains high levels of protein expression. Bac-to-Bac® relies on generation of recombinant baculovirus by site-specific transposition in E. coli rather than homologous recombination in insect cells. The Bac-to-Bac® Baculvirus Expression System is highly regarded in academic Literature. Approximately 100 citations every year since 2000!

Traditional baculovirus systems require purification and amplification of an initial low-titer viral supernatant. This requires a time-consuming plaque assay. Bac-to-Bac®'s pFastBac™ vector, however, recombines with the parent bacmid in DH10Bac™ E. coli competent cells to form an expression bacmid. Transfect the bacmid into insect cells for fast production of a high titer of pure recombinant baculovirus particles in the very first transfection. You'll save weeks of precious time. Collect a pure P2 baculovirus stock on Day 10 without the necessity of tedious plaques assay (See Figure 1)

High expression, easy screening
pFastBac™ uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell lines such as Sf9, Sf21, and High Five™ cells.

New additions:
Bac-to-Bac®'s pFastBac vectors are now available as pFastBacTOPO® vectors too. pFastBacTOPO® vectors are Blunt® TOPO® vectors and are supplied with Mach1™-T1R E. coli for easy cloning and pFastBac⁄GOI propagation. The new vectors enable you to:

• Amplify your gene of interest (GOI) with a PCR enzyme of highest fidelity such as Accuprime™ Pfx SuperMix.

• TOPO® clone the blunt-end PCR product into the new vector in only 5 minutes!

• Visualize colonies 8 hours after plating on ampicillin selective plates because Mach1™-T1R E. coli cells have a faster doubling time compared to other standard cloning strains.

• With the Bac-to-Bac® C-His TOPO® cloning or expression kit, you can produce a C-terminal His-fusion protein with a TEV cleavage site to purify with nickel-chelating resins (Invitrogen’s ProBond™ Purification System) and generate a native protein with the aid of Invitrogen’s popular AcTEV™ Protease. This is the Invitrogen’s only vector that contains a C-terminal TEV cleavage site!

The new vectors combine the TOPO® cloning technology with the highly regarded Bac-to-Bac® baculovirus expression features to enable easy cloning and high-level protein expression.
The pFastBacTOPO® vectors are offered as cloning and expression kits.

MembraneMax™ HN Protein Expression Kit (Invitrogen™)

MembraneMax™ HN Protein Expression Kits delivers high yields of soluble (dispersed) membrane proteins using the MembraneMax™ reagent, which is an planar phospholipid membrane bilayer surrounded by a scaffold protein (also called a nanolipoprotein particle or NLP) that also offers convenient His-tag purification. Combining this technology with a scalable cell-free expression E. coli extract delivers microgram to milligram quantities of your membrane protein specifically for high throughput expression screening, expression of membrane proteins that would otherwise be toxic in cell-based systems and functional or activity assays. With high uniformity and consistent structure, MembraneMax reagent minimizes formation of membrane protein aggregates or clumping often caused by inconsistent size, shape and structure typical observed with microsomes or micelles. The MembraneMax™ Protein Expression kits contain all the necessary reagents for successful expression of your membrane protein.
Key Advantages of the MembraneMax™ Protein Expression Kits:
• Optimized cell-free expression delivers high yield of membrane protein from nanogram to milligram quantities
• MembraneMax™ reagent enables a monodispersed population of soluble membrane protein-NLP complexes
• Convenient His-tag allows for simple purification of native or un-tagged membrane proteins
• Expression format amenable to high-throughput protein synthesis for screening and expression of toxic proteins
• Production of a homogeneous population of your membrane protein
• Convenient kit format includes the necessary reagents for protein expression—simply add your gene of interest to get started

Jump In™ T-REx™ HEK 293 Kit

The Jump-In™ T-REx™ HEK293 Kit allows the targeted integration of genetic material into a specific pre-engineered R4 site in the Jump-In™ HEK293 cell line, to create an inducible isogenic stable cell line with less effort and in less time than traditional cell engineering methods. These cells stably express the tetracycline repressor protein, allowing for inducible expression of your retargeted gene of interest upon addition of doxycycline.

The high retargeting efficiency, made possible by the R4 sites in the HEK293 cell line, allows for the use of isogenic pools for additional experiments without the need for clonal selection. Alternatively, the high retargeting efficiency allows for the easy selection of a positive stable clone expressing your gene of interest.

The Jump-In™ T-REx™ HEK293 Kit lets you:

• Quickly and efficiently develop stably engineered isogenic cell pools in about half the time compared to traditional cell engineering methods
• Inducibly express your gene of interest using doxycycline
• Utilize isogenic expression from a defined genomic locus as the ideal solution for comparative analysis of gene families, isoforms, or orthologs
• Generate multiple cell lines in parallel using the simplified work flow
• Easily access the technology without complicated licenses or restrictions to interpret

Save Time with Rapid and Efficient Generation of Engineered Cell Lines
With the Jump-In™ T-REx™ HEK293 Kit you can generate functional cell pools in as little as 2 weeks without laborious clone isolation and analysis, and the streamlined protocol makes it easier to generate several cell lines at the same time. Even generation of clonal cell lines can be done with reduced time and effort due to the high percentage of positive clones. In addition, the Jump-In™ technology gives you the freedom to generate an unlimited number of cell lines without restrictive licensing requirements.

Expand Your Experimental Capabilities
The Jump-In™ T-REx™ HEK293 Kit allows for inducible expression of your toxic or unstable gene of interest and is the ideal solution for targets and assays where transient engineering technologies are problematic. The kit also provides a convenient way to create target panels of gene families, isoforms, or orthologs. Genes coding for large proteins or multi-unit proteins are not a problem since the Gateway® destination vectors accept large inserts.

The kit includes:

Jump-In™ T-REx™ HEK293 cells (2 vials @ 1 ml each)
pJTI™ R4 Dest CMV-TO pA vector (100 µg)--A Gateway® Destination vector that drives inducible expression of your gene of interest under control of the strong CMV promoter by inclusion of the Tet-Operon.
pJTI™ R4 Int vector (100 µg)--Expresses the R4 integrase to facilitate the retargeting of your gene of interest into the R4 site.

Sold separately:

pJTI™ R4 CMV-TO MCS pA vector--A multi-site cloning vector that drives inducible expression of your gene of interest under control of the strong CMV promoter by inclusion of the Tet-Operon.
pJTI™ R4 EXP CMV-TO EmGFP pA vector--A control vector with inducible expression of GFP under control of the strong CMV promoter by inclusion of the Tet-Operon.

MembranePro™ Functional Protein Support Kit (Invitrogen™)

For customers studying GPCRs and other membrane proteins, MembranePro™ is the ready-to-use system that delivers enriched, functional membrane proteins efficiently and reliably. Benefits of MembranePro™ include:

• Proteins are displayed on human (or other mammalian) cell membranes
• Cellular quality control and mammalian posttranslational processing helps produce functional protein
• Proteins bud off as lipoparticles and are easily collected from the culture medium
• Lipoparticles are enriched with expressed receptors

MembranePro™ workflow is less labor intensive in comparison to traditional production of membrane fractions. Briefly, the MembranePro™ protocol allows you to:

1. Clone your gene of interest using a TOPO® vector
2. Transfect with Lipofectamine™ 2000 and MembranePro™ Reagent into 293FT cells
3. Harvest lipoparticles 48h post-transfection, concentrate with MembranePro™ Precipitation Mix
4. Store lipoparticles at -80 C or proceed to downstream assays

MembranePro™ products are offered in two configurations – as an expression kit (TOPO® vector, Lipofectamine™ 2000, MembranePro™ Precipitation Mix, MembranePro™ Reagent, 293FT cells) and as a support kit (Lipofectamine™ 2000, MembranePro™ Precipitation Mix, MembranePro™ Reagent). The expression kit is only available in a 10 reaction size; the support kit is available in 10, 60 and 600 reaction sizes.

Retic Lysate IVT™ Kit with Manual (Invitrogen™)

The Retic Lysate IVT™ Kit efficiently translates in vitro-synthesized transcripts, poly(A) RNA, and total RNA, including difficult-to-translate RNAs. The optimized lysate results in high protein yield, biological activity, and consistent performance. Advantages of the Retic Lysate IVT™ Kit:

• Efficiently translates a wide variety of high molecular weight proteins and both capped and uncapped mRNAs
• Accommodates large volume of template mRNA

How it works
The lysate has been optimized for translation by the addition of an ATP-regenerating system, hemin, and calf liver tRNA. Included in the kit is an optimized Translation Mix (-Met or -Leu) for the translation of both capped and uncapped messages along with High and Low Salt buffers. These buffers can be used to maximize protein yield and activity from difficult-to-translate sequences. A report in Nucleic Acids Research (1) found that some commercial reticulocyte lysates had poor fidelity for initiation at the appropriate AUG initiator codon. The Retic Lysate IVT™ Kit was tested using Kozak's constructs and it demonstrated high fidelity for initiating at the proper AUG.

Reference
1. Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res 18:2828.

pTrcHis TOPO™ TA Expression Kit (Invitrogen™)

The pTrcHis- and pTrcHis2-TOPO® TA Expression Kits allow fast, efficient cloning of PCR products into a prokaryotic expression vector for high-level, regulated expression from the trc promoter. The vector in each kit, pTrcHis-TOPO® or pTrcHis2-TOPO®, includes a minicistron element for enhanced translation efficiency of eukaryotic proteins in E. coli. Both vectors are provided linearized and activated with topoisomerase I for 5-minute TOPO® Cloning with >85% cloning efficiency. To simplify protein purification and detection, the pTrcHis-TOPO® and pTrcHis2-TOPO® vectors include the following features:

pTrcHis-TOPO®

N-terminal Xpress™ epitope for detection with an Anti-Xpress™ Antibody
• N-terminal polyhistidine (6xHis) tag for purification using nickelchelating resin and detection with an Anti-HisG Antibody
• Enterokinase cleavage site for efficient removal of N-terminal fusion tags

pTrcHis2-TOPO®
C-terminal c-myc epitope for detection with an Anti-myc Antibody
• C-terminal polyhistidine (6xHis) tag for purification using nickel-chelating resin and detection with an Anti-His(C-term) Antibody

MAX Efficiency™ Transformation Reagent for Algae (Invitrogen™)

MAX Efficiency® Transformation Reagent for Algae is a buffer that enhances transformation efficiency for multiple strains of Chlamydomonas species. One of the biggest hurdles in research and development with Chlamydomonas has been the introduction of exogenous DNA into Chlamydomonas strains due to rigid cell walls. Methods such as glass bead agitation, electroporation, and microparticle bombardment are available but often result in low transformation efficiency.

MAX Efficiency® Transformation Reagent for Algae increases the permeability of the Chlamydomonas cell wall and facilitates increased delivery of DNA into the cell nucleus by electroporation. Simply grow the Chlamydomonas cells to early log phase, harvest by centrifugation, and wash twice with MAX Efficiency® Transformation Reagent prior to electroporation. To date, we have seen >200-fold increase in transformation efficiency over previously recommended conditions in 10 different Chlamydomonas strains, including wild type and mutants, using circular or linear DNA, as well as PCR fragments.

• Obtain >1000 transformants/µg DNA for cell wall(+) strains
• Obtain >100 transformants/µg DNA for cell wall(-) strains
• Transform circular or linear DNA or PCR fragments

Bac-to-Bac™ N-His TOPO™ Cloning Kit (Gibco™)

Bac-to-Bac® Baculovirus Expression is an efficient method for producing baculovirus for high-level protein expression in insect cells. It relies on generation of recombinant virus by site-specific transposition in E. coli rather than homologous recombination in insect cells. New: pFastBacTOPO® vectors!

Reliable and fast protein expression
The Bac-to-Bac® Baculovirus Expression System is faster and easier than traditional baculovirus expression, and it maintains high levels of protein expression. It is based on site-specific transposition in E. coli rather than homologous recombination in insect cells. The Bac-to-Bac® Baculvirus Expression System is highly regarded in academic Literature. At least 80 citations every year since 2000!

Traditional baculovirus systems require purification and amplification of an initial low-titer viral supernatant. This requires a time-consuming plaque assay. Bac-to-Bac®'s pFastBac™ vector, however, recombines with the parent bacmid in DH10Bac™ E. coli competent cells to form an expression bacmid. Transfect the bacmid into insect cells for fast production of a high titer of pure recombinant baculovirus particles in the very first transfection. You'll save weeks of precious time. Collect a pure P2 baculovirus stock on Day 10 without the necessity of tedious plaques assay (See Figure 1)

High expression, easy screening
pFastBac™ uses the strong polyhedrin promoter to generate high levels of expression in a variety of insect cell lines such as Sf9, Sf21, and High Five™ cells.

New additions:
Bac-to-Bac®'s pFastBac vectors are now available as pFastBacTOPO® vectors too. pFastBacTOPO® vectors are Blunt® TOPO® vectors and are supplied with Mach1™-T1R E. coli for easy cloning and pFastBac⁄GOI propagation. The new vectors enable you to:

• Amplify your gene of interest (GOI) with a PCR enzyme of highest fidelity such as Accuprime™ Pfx SuperMix.

• TOPO® clone the blunt-end PCR product into the new vector in only 5 minutes!

• Visualize colonies 8 hours after plating on ampicillin selective plates because Mach1™-T1R E. coli cells have a faster doubling time compared to other standard cloning strains.

• With the Bac-to-Bac® N-His TOPO® cloning or expression kit, you can produce a N-terminal His-fusion protein with a TEV cleavage site to purify with nickel-chelating resin (Invitrogen’s ProBond™ Purification System) and generate a native protein with the aid of Invitrogen’s popular AcTEV™ Protease.

The new vectors combine the TOPO® cloning technology with the highly regarded Bac-to-Bac® baculovirus expression features to enable easy cloning and high-level protein expression.
The pFastBacTOPO® vectors are offered as cloning and expression kits.

NativePure™ Mammalian Affinity Purification Kit (Invitrogen™)

The NativePure™ Mammalian Affinity Purification Kit provides an easy-to-use, efficient method for purifying native protein complexes.
NativePure™ pcDNA™ Gateway® vectors (Figure 1) are designed to allow expression of biotinylated recombinant fusion proteins in mammalian cells through N- or C- terminal fusion of your recombinant protein of interest to the capTEV™ Tag.
The capTEV™ Tag consists of a BioEase™ in vivo biotinylation peptide, two Tobacco Etch Virus (TEV) protease recognition sites, and a 6XHis tag. The BioEase™ tag, a 72-amino acid sequence from K. pneumoniae, directs in vivo biotinylation of a specific lysine residue.
The strong interaction of biotin and streptavidin is used to purify the protein complexes under native conditions using Streptavidin Agarose included in the NativePure Affinity Purification Kit. Biotinylated proteins and protein complexes remain bound, while impurities are removed by thorough washing of the Streptavidin Agarose column.
Two tandem AcTEV™ Protease recognition sites facilitate the release of bound biotinylated proteins and protein complexes from Streptavidin Agarose, creating a useful strategy for protein purification.
Following purification, the protein complexes are ready for analysis in many downstream applications including SDS-PAGE, western analysis, mass spectrometry, and native electrophoresis using the NativePAGE™ Novex® Bis-Tris Gel System.

The NativePure™ Mammalian Affinity Purification Kit provides:
• Gateway® vectors designed to express your protein of interest in multiple expression systems
• pcDNA™ vectors with the capTEV™ Tag that allow in vivo biotinylation of your protein of interest
• Streamlined purification protocols with Streptavidin Agarose

Contents and Storage:

The NativePure™ Mammalian Affinity Purification Kit includes the NativePure™ pcDNA™ Gateway® Vector Kit and the NativePure™ AffinPurification Kit. The NativePure™ pcDNA™ Gateway® Vector Kit and NativePure™ Affinity Purification Kit are also available separately. The NativePure™ pcDNA™ Gateway® Vector Kit consists of the pcDNA™3.2/capTEV™-NT/V5-DEST vector (N-terminal purification tag), the pcDNA™3.2/capTEV™-CT/V5-DEST vector (C-terminal purification tag), and the pcDNA™3.2/capTEV™-NT/V5-GW/ARPC2 control vector. The NativePure™ Affinity Purification Kit contains Streptavidin Agarose, NativePure™ 5X Lysis/Binding Buffer, NativePure™ 10X TEV Cleavage Buffer, 100 mM DTT (dithiothreitol), 10% NP40, AcTEV™ Protease, NativePure™ Columns, and NativePure™ Concentrators. Store Streptavidin Agarose, 10% NP40, NativePure™ 5X Lysis/Binding Buffer, NativePure™ 10X TEV Cleavage Buffer, NativePure™ Columns, and NativePure™ Concentrators at +4°C. Store the NativePure™ pcDNA™ Gateway® Vector Kit, AcTEV™ Protease, and 100 mM DTT at -20°C. All components are guaranteed stable for 6 months when properly stored.