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Reconstituted Cell-free Protein Synthesis System, PURESYSTEM - General Questions


PURESYSTEM Top

products_bco_20090714_01.jpgReconstituted Cell-free Protein Synthesis System@<PURESYSTEM®> Flyer PDFPDF

General Questions

Q1. What are the advantages of PURESYSTEM, compared with other cell-free kits?
A1.
  • PURESYSTEM is "PURE" as shown by its name, because it is a reconstituted system containing only defined molecules.
  • Easy and fast protein synthesis as well as purification.
  • Is able to form disulfide bond(s).
  • No special device required.
  • Is able to use a PCR-amplified DNA as a template
  • Molecular chaperons are available for production of an active form of protein
  • Service of optimization of template DNA available
  • Free technical-service for all users
  • Custom made PURESYSTEM kits with varying components are available......and more!
Q2. Who would be recommended to use PURESYSTEM?
A2.
  • Customers performing experiments of protein expression using bacteria, yeast, animal cells, etc.
  • Customers requiring less than 10 mg of protein
  • Customers synthesizing a large number of proteins for assays (ex. Improving activity or stability of enzyme by mutation, modifying therapertic proteins by mutation)
  • Customers labeling proteins
  • Customers preparing protein antigen
  • Customers using other cell-free kits
Q3. What kind of customers are NOT recommended?
A3.
  • Customers who have a target protein which a sugar chain is needed for revitalization.
  • Customers who needs the protein in unit of grams.
Q4. How much will be the amount of protein synthesized by PURESYSTEM?
A4. About 100mg/ml.
Q5. Is it possible to synthesize membrane protein?
A5. As well as other cell-free kits, synthesis of complex protein will be difficult. It is possible to synthesize polypeptide without the membrane penetrating portion.
Q6. Is it possible to modify the protein after translation?
A6. We were the first among others to develop the cell-free kit enabling disulifide-bonded proteins(PURESYSTEM S-S), but to synthesize protein with sugar chain, phosphorylated proteins etc. are not possible by PURESYSTEM. These modification inside the animal body will be also difficult by using cells.

Template

Q1. Can I synthesize proteins containing tags in PURESYSTEM?
A1. Yes, all tags except histidine-tag can be used in PURESYSTEM.
Q2. What can be used as a template in PURESYSTEM?
A2. PCR product, plasmid DNA, and mRNA can be used as a template.
Q3. What is the method for preparing a plasmid DNA?
A3. Plasmid DNA can be isolated by any commercially available kits.
We recomend a Plasmid Mini Kit (Qiagen) of them.
Q4. Which termination codon can be used?
A4. All the stop condon (TAG, TGA and TAA) can be used in PURESYSTEM classic II.

Protein Synthesis

Q1. What is the yield of the synthesized protein in PURESYSTEM?
A1. The yield is dependent on each protein. To give one example, the yield of DHFR is 50 ug/ml.
Q2. What is the protein size range that you can synthesize in PURESYSTEM?
A2. From tripeptide to protein of approximately 100 kDa. Domain expression of much larger protein is also possible.
Q3. Can I synthesize proteins containing tags in PURESYSTEM?
A3. Yes, all tags except histidine-tag can be used in PURESYSTEM.
Q4. Do proteins synthesized in PURESYSTEM show biological activity?
A4. Yes / No. Again, it depends on the protein, but we have successfully synthesized DHFR with an enzyme activity
Q5. What can be used as a template in PURESYSTEM?
A5. PCR product, plasmid DNA, and mRNA can be used as a template.
Q6. Which termination codon can be used?
A6. All the stop condon (TAG, TGA and TAA) can be used in PURESYSTEM classic II.
Q7. The control protein (DHFR) cannot be detected.
A7. Please verify that the expiration date and storage temperature of the kit is as recommended. If there is no problem with those conditions, please repeat the experiment taking care to keep the experiement RNase-free at each step.
A8. Only the control protein can be detected.
Q8. Please check the sequence, purity and concentration of your template DNA.
A9. The target protein can be detected but the yield is very low.
Q9. The yield is dependent on the target protein. When the yield is low, please examine the reaction conditions such as extension of incubation time or varying the amount of template DNA.
A10. Are there any alternative methods of purification?
Q10. For example, ion-exchange column and/or gel filtration can be used.

 

Purification

Q1. Are there any alternative methods of purification?
A1. For example, ion-exchange column and/or gel filtration can be used.

 

Detection

Q1. The control protein (DHFR) cannot be detected.
A1. Please verify that the expiration date and storage temperature of the kit is as recommended. If there is no problem with those conditions, please repeat the experiment taking care to keep the experiement RNase-free at each step.
Q2. Only the control protein can be detected.
A2. Please check the sequence, purity and concentration of your template DNA.
Q3. The target protein can be detected but the yield is very low.
A3. The yield is dependent on the target protein. When the yield is low, please examine the reaction conditions such as extension of incubation time or varying the amount of template DNA.