Navigation Links
Translate High Yields of Biologically Active Proteins

ActivePro In Vitro Translation Kit


Cell-free translation systems are a convenient method for synthesizing proteins in vitro. The major advantages of these in vitro translation systems over conventional in vivo systems are greater flexibility, faster reaction times, and less labor-intensive protocols.

Ambions ActivePro In Vitro Translation Kit is a cell-free, E. coli based translation kit that excels at expressing a wide range of functional proteins for a variety of downstream applications. ActivePro generates high yields of proteins with higher biological activity compared to other commercially available kits. This report demonstrates that a number of complex human proteins can be efficiently translated using this technology.


Performance with Human Templates
Thirteen human full length, open reading frame (ORF) clones were selected from the NIH Mammalian Gene Collection (MGC). The corresponding full length sequences were subcloned into Ambions optimized translation vector (included in the ActivePro Kit) and sequences were confirmed. 12 g each of the 13 constructs were translated in a standard 50 l ActivePro reaction. Each reaction included 35S-labeled methionine and was incubated for 1 hour at 37C. Protein yields, as determined by TCA precipitation, were calculated based on the number of labeled methionines in each sequence. Figure 1 lists the genes translated, the corresponding protein molecular weights, and the protein yields. While yields varied with the translated protein, in all but one case, 1030 g of protein were produced per reaction.

Figure 1. Expression of 13 Human Proteins Using the ActivePro Kit. DNA templates (12 g) for 13 human proteins were in vitro transcribed/translated in duplicate using the ActivePro Kit and 20 Ci 35S-labeled methionine. A 5 l sample from each reaction was used to calculate yields by TCA precipitation.


Large Scale Reactions with Human Templates
To demonstrate ease of scalability using the ActivePro Kit, reaction volumes ranging from 50 l to 2 ml were assembled and incubated for 1 hr at 37C. Two proteins, caspase 3 and MAPKinase 9, were synthesized in these scaled-up reactions and yields were calculated (Figure 2).

Figure 2. ActivePro Scale-Up Reactions. Caspase 3 and MAPKinase9 DNA templates were transcribed/translated using ActivePro in a range of reaction sizesfrom 50 l to 2 ml. (A) Quantitated protein yields as determined by TCA precipitation. (B) Autoradiograph of the 35S-labeled proteins.


In one hour, several hundred micrograms of protein were obtained in the 1 ml reaction. Nearly 1 mg of human protein was obtained for MAP kinase in the 2 ml reaction. The sharp bands seen in Figure 2 illustrate how the quality of full length protein is not compromised in these scaled-up protocols.


Protein Expression With ActivePro Surpasses Competitors
To compare the efficiency of ActivePro with two competing systems, five human templates were expressed using the manufacturers protocols. Both yields (Figure 3) and biological activities of the reaction products (Figure 4) were assayed. ActivePro gave the highest yields for all five of the proteins translated. And in all but one case (DHFR), ActivePro-translated proteins showed the highest enzymatic activity.

Figure 3. Comparison of Protein Yields Across E. coli Lysate Kits. DNA templates for 5 human proteins were in vitro transcribed/translated using Ambions ActivePro Kit and two other commercially available kits, according to protocol. The proteins analyzed were dihydrofolate reductase (DHFR), adenylate kinase 1 (AK1), carbonic anhydrase 2 (CA2), caspase 3 (CASP3), and casein kinase 1 (CK1). (A) Quantitated protein yields as determined by TCA precipitation. (B) Autoradiographs of the 35S-labeled proteins.

Figure 4. Comparison of Biological Activity Across E. coli Lysate Kits. The proteins as produced from Figure 3 were tested in functional assays. Relative activities from protein preparations synthesized using two commercially available kits were compared to that from the ActivePro Kit. Dihydrofolate reductase (DHFR): Activity was measured with a standard DHFR assay, which follows the rate of conversion of dihydrofolate and NADPH to tetrahydrofolate and NADP+ through the successive decrease of absorbance of NADPH at 340 nm at 30C [1]. Adenylate kinase 1 (AK1): Activity was measured by a luciferase-based ATP bioluminescence assay (Sigma) in the presence of 1 mM ADP [2]. Carbonic anhydrase 2 (CA2): Esterase activity of CA2 was monitored by the net increase in absorbance at 348 nm in a p-nitrophenyl acetate hydrolysis assay [3]. Caspase 3 (CASP3): Catalytic activity was determined by measuring the proteolytic cleavage of the fluorogenic substrate Ac-DEVD-AFC for 1 hr in a 96 well plate reader (400 nm excitation wavelength, 505 nm emission wavelength). Casein kinase 1 (CK1): Activity was determined by phosphorylation of casein kinase 1 substrate peptide (Upstate).


Summary
ActivePro is a superior, cell-free in vitro translation kit delivering both high yields and active protein. It is fast, easy to use, and scalableas much as 1 mg of protein can be synthesized in just 1 hour. Compared to similar kits, ActivePro consistently expresses more biologically active protein in extremely high yields. Because it does not require special equipment, it is the fastest and most economical kit for producing milligram quantities of protein suitable for any downstream application.


back to top


Ordering Information
Cat# Product Name Size 1295 ActivePro In Vitro Translation Kit 25 rxns
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Translate High Yields of Active Protein
2. Translate More Protein with Higher Biological Activity
3. Yeast Protein Production System Features High Yields and One-Step Purification
4. ArrayScript RT: Maximize Yields of Full-length cDNA
5. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
6. Synthesize High Yields of Biotinylated aRNA
7. Production of Full-Length, Biologically Active MEK1WT and MEK1CA
8. Highly Active and Removable AffinityTagged Lambda Phosphatase
9. Rapid PCR* Using Active Tube Temperature Control On Thermos PCR Machines
10. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
11. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Translate High Yields Biologically Active Proteins

(Date:9/30/2014)... Calif. , Sept. 30, 2014 FemCap ... women,s health announces its receipt of a patent No: ... menstrual cup has also received FDA clearance in the ... and the CE in Europe. This patent is related to ... with this issuance, the company is releasing FemmyCycle Teen, ...
(Date:9/30/2014)... 30, 2014 Lyme Research Alliance ... Lyme disease research at universities, today announced the awarding ... the identification, treatment and cure for Lyme and other ... the most applications in its history—over 20 grant proposals—a ... we were pleased to receive so many solid applications ...
(Date:9/30/2014)... Georgetown, Mass. (PRWEB) September 30, 2014 ... components, products, and specialty packaging has recently introduced ... BioShell® Suspension Pack . The new insulated ... the cold chain distribution process. UFP Technologies’ BioShell ... use biopharmaceutical bags during storage, handling and shipping. ...
(Date:9/29/2014)... Each year in the US, ... attention. In the military, burn injury is a ... medical care. More than 800 service members sustained ... heal slowly, remain inflamed and often become infected, ... and functionally damaging. While developments in supportive care ...
Breaking Biology Technology:The FemmyCycle 2Lyme Research Alliance Awards Nearly $600,000 in New Grants 2Lyme Research Alliance Awards Nearly $600,000 in New Grants 3Lyme Research Alliance Awards Nearly $600,000 in New Grants 4UFP Technologies Introduces Insulated Shipping Container For Bulk Drug Transportation 2UFP Technologies Introduces Insulated Shipping Container For Bulk Drug Transportation 3Faster Healing with Fewer Scars 2
... wireless bids ,The City of Madison received bids from ... InSite Wireless to build wireless hotspots around the airport ... Monday. The winning vendor will pay the city, county and ... able to charge access fees. Certain local sites could be ...
... OpGen , a company that makes ... individuals, has completed the final stage of its $5 ... on University of Wisconsin-Madison technology, already sells its testing ... will be split between marketing that service and new ...
... that word? It was as popular in Web circles in the ... long and short of it was that the Web was going ... to consumers, either in business or retail. , ,Everybody still in ... would get better prices, and so would consumers. A real win-win ...
Cached Biology Technology:Tech Digest: Madison wireless, angels, Third Wave, Esker, Research Park 2OpGen completes $5 million investment round 2A funny thing happened on the way out of the back office 2A funny thing happened on the way out of the back office 3
(Date:9/29/2014)... beginning of the age of dinosaurs, gigantic reptilesdistant relatives ... and others in water and it was thought they ... University of Tennessee, Knoxville, researcher in the thigh of ... , Stephanie Drumheller, an earth and planetary sciences ... Sterling Nesbitt examined 220-million-year-old bite marks in the thigh ...
(Date:9/29/2014)... Growing Demand From Huge ... Security Concerns Continue to Propel Biometrics Market in ... is one of the fastest growing biometrics markets ... majorly in various government projects such as National ... biometrics dominates the country,s biometrics market, with other ...
(Date:9/29/2014)... Oktoberfest is here, and that means ... for a cold one, taking that first ... chemicals have reacted to form compounds that ... Reactions explains why beers get skunky, and ... brews from going bad. Learn all about ...
Breaking Biology News(10 mins):Tooth serves as evidence of 220 million-year-old attack 2India Biometrics Market to Surpass USD2.7 Billion by 2019, Finds TechSci Research Study 2India Biometrics Market to Surpass USD2.7 Billion by 2019, Finds TechSci Research Study 3
... 100 scientists worldwide publish joint study in Science , ... the US have been probing the genome of the ... results in some aspects have completely overturned geneticists' traditional ... Science on 2nd September. The general conclusion of the ...
... carry and spread to humans is the focus of ... distinguished professor of biochemistry and head of the department ... how insects protect themselves against infection. They think the ... researchers have made progress in understanding which molecules are ...
... the latest issue of Restoration Ecology finds that in ... Oyster reefs are self-sustaining, and are additionally attractive because ... growth, and contribute to overall ecosystem stability and quality. ... stimulate the oysters' settlement; reefs are therefore able to ...
Cached Biology News:Mouse genome much more complex than expected 2K-State researchers study insects' immune system 2
Vesicle Associated Membrane Protein (VAMP 1/2)...
...
Anti-OSMR Monoclonal Antibody Research Focus: cytokine/growth factor Storage: 4C Shipping Temperature: 4C...
ROCK-1 (cleaved) Monoclonal Ab 100 ug purified mouse monoclonal antibody. Clone# 12M03. Reacts with human. Tested in Western blot analysis....
Biology Products: