Excellgen's scientists have average of 15 years experience in molecular biology, protein expression and purification. Our team is proficient in molecular cloning, producing and purifying of recombinant proteins in a variety of expression systems including cell-free protein synthesis, E. Coli, yeasts, baculovirus/insect cells and mammalian cells (CHO and 293). We have ÄKTAExplore and ÄKTAPurifier FPLC systems, ~ 30 large GE Pharmacia prepacked columns (gel filtration, hydrophobic, ion exchange, desalting) and numerous affinity columns. By combining state-of-the-art fementation and ÄKTA FPLC chromatography systems, we can produce quantities of pure recombinant proteins ranging from miligrams to a few grams.
This package is designed for producing and purification of 3 mg recombinant protein in Baculovirus Expression System.
| Timeline |
Milestones |
Tasks |
| 1-2 weeks |
Cloning and Subcloning |
- PCR amplification using high fiderity DNA polymerase
- Clone PCR product into an expression vector
- Restrition digestion analysis and sequencing of the construct to ensure correct sequence and frame
- Preparation of plasmid DNA
|
| 1 week |
Generation of
Bacmid |
- Transform DH10Bac competent cells with plasmid DNA
- Selection of Bacmid clones containing the gene of interest
- Purification of Bacmid DNA
|
| 2-3 weeks |
Baculovirus Generation and
Amplification |
- Generation, identification of baculoviral colonies expressing target proteins
- Plaque purification of baculoviral colonies expressing target proteins
- Amplification of baculovirus for high titer stock
- Titration of the baculovirus
|
| 1 week |
Protein Expression |
- Infect insect cells with the baculovirus
- Culture the insect cells
- Centrifuge and collect both supernatant and cell pellet
|
| 1 week |
Protein Purification |
- Purification using an affinity column
- Further Purification using AKTA FPLC systems
- Dialysis and concentrate protein
- SDS-PAGE or Western blot analysis of the purified protein
- Determination of protein concentration
- Delivery of purified and ready-to-use protein
|
Total time required: 6 to 8 weeks.
Deliverables
- Minimum 3 mg purified recombinant protein, > 95% purity
- QC Data
- Chromatograms
Required Materials
- 5 µg of plasmid containing gene of interest plus complete vector sequence in electronic format
- or protein sequence for gene synthesis (with additional price for gene synthesis)
- or accession number so we can buy cDNA clone from suppliers such as ATCC, Open Biosystems, Origene, GeneCopoeia
Quotation and Ordering
To request a quotation, please request a quotation form from customer services crm@excellgen.com
To order, please send the completed quotation form to us by email or fax with a formal PO (Purchase Order) or credit card information. You can also submit PO/credit card information by phone.
If submitting samples, please mail them together with a hard copy of the completed quotation form to: Protein Services, Excellgen, 847 Quince Orchard Blvd, Suite H, Gaithersburg, MD, 20878, US.
800-338-6318
