SHEATECH, INC
Gene Expression and Proteomics

FAQs & Protocols

FAQs


1) What are the main differences between E. coli expression and Baculovirus expression? 
Baculovirus expression is an eukaryotic expression system with post-translational modification during protein expression whereas in E.coli is a prokaryotic expression system without post-translational modifcation. Eukaryotic genes can produce soluble recombinant protein in Baculovirus expression system. 

2) How do you grow healthy sf9 cells? 
It is imperative that you choose the right medium and incubation condictions for the insect sf9 cells. Make sure you grow the sf9 cells in log phase and low passages free of overgrowth and with the proper amount of dilution. Check to make sure that the monolayer is free of enlarged cells as well as floating cells. 

3) What type of medium does insect cells grow in? 
Insect cells can grow in serum free medium and/or complete medium (TNM-FH, 10% FCS). It is important to grow sf9 cells in log phase with serum free or complete medium. It usually takes several passages for the insect cells to adapt to the serum free medium. Once insect cells adapt and grow in serum free medium, they can grow well in monolayer culture and suspension culture in a spinner flask. 

4) How do you prevent contamination of co-transfection? 
Be sure to confirm transfection reagent and medium with your vendor. Make sure that the DNA sampleis suspended in sterile TE buffer. Add suitable antibiotic to the medium for co-transfection.

5) Why has co-transfection failed to produce recombinant virus? 
Call vendor to make sure that the lot of virus DNA is indeed suitable for co-transfection. Check the condition and density of seeding sf9 cells. If sf9 cells are seeded too densely, then they might generate low titer virus during co-transfection. The virus titer from the co-transfection may be too low to identify the infected sf9 cells. By using virus amplification, the insected sf9 cells can be identified by microscope. 

6) Why aren't there any plaques on the plaque assay? 
Make sure the sf9 cells are healthy and suitable for plaque assay. Be sure to check the density. Virus titer may be too low or too high to be detected on the plaque assay. In general, four serial dilutions of virus should be enough to cover the broad range of virus titer. Ensure that the temperature of agarose layer for plaque assay is appropriate. Be sure to prepare fresh Neutral red solution for each plaque assay. 

7) How do you generate high-titer virus stocks? 
Start from a plaque and amplify titer from a small volume to a large volume using sf9 cells. Make sure you infect healthy sf9 cells for high titer virus. 

8) How is the stability of the high titer virus? 
High titer virus is stable in FCS medium for a couple of months. Virus titer can be determined by plaque assay or end point dilution. Confirm virus titer first and infect healthy sf9 cells for protein expression. 

9) How do you prevent cracks on the plaque assay and allow plaques to grow around the center region of the plate? 
Be sure to add 1mL of medium once agarose layer has set. The liquid medium prevents cracks on agarose layer during incubation. Seed healthy sf9 cells uniformly in the dish, add diluted virus solution, and gently remove the virus completely; by doing so, the plaques will locate uniformly in the dish. 

10) Why is it so important to optimize conditions for protein expression? 
Both recombinant virus and recombinant protein might affect protein expression in Baculovirus expression system. A better and higher yield will result under optimized conditions. Both MOI and insect cells lines are used to find out optimized conditions. 

11) How much protein will be produced from one-liter insect cell expression? T
he amount of protein produced will range from 10mg/liter to 100ug/liter; this depends on the genes and the growing conditions. Most of the proteins are soluble in the Baculovirus expression system; however, some proteins are insoluble in this system. Also, neutral detergent may help the solubility of recombinant proteins. 


*Please Note: Sheatech, Inc. has provided the above questions and short answers/suggestions to these common problems. If you are, however, facing an unusual issue or problem not listed above, please feel free to contact Sheatech, Inc. at (619) 788-8188 or email us at info@sheatechinc.com. We will work hard to help you resolve this problem.







DNA Protocols

   

    Mini-prep/Midi-prep
    Agarose Gel Electrophoresis
    Agarose Gel Extraction
    Phosphorylation of Oligomers
    Ligation/Transformation
   



Protein protocols

   

    His-Protein Purification with Ni-NTA Column
    SDS-PAGE
    Western Blot




Other protocols


    Insect Cells Culture