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View the relevant questions below:
Here are the most common possibilities/remedies for why your protein may not be expressed:
- Low transfection efficiency: Optimize your transfection by performing stable selection or use methods that permit examination of individual cells. You can also try to increase the level of expression by changing the promoter.
- Detection method used may not be able to detect expression within the cell: Optimize the detection protocol or find more sensitive methods.
- Protein may be truncated or degraded: Check by Northern blotting.
- Possible time-course issue: Expression of a protein will fluctuate over time depending on the nature of the protein; therefore, a time course for expression is recommended.
- Possible solubility issue: Overexpression in E. coli may lead to the formation of inclusion bodies that are not solubilized with standard SDS-PAGE sample buffer and boiling; to check the insoluble fraction, it may be necessary to use 6–8 M urea and sonication.
- Possible cloning issues: Verify clones by restriction digestion and/or sequencing; ensure proper expression elements are present.
Here are the most common possibilities/remedies for why your antibiotic resistant clones are not expressing your gene of interest:
- Detection method may not be appropriate or sensitive enough.
- Insufficient number of clones screened.
- Inappropriate antibiotic concentration used for stable selection (make sure kill curve was performed correctly); for stable selection, the location of integration of your gene of interest is important and can cause differing levels of expression.
- Expression of gene product (even low level) may not be compatible with growth of the cell line (i.e., your gene is toxic). Therefore, try using an inducible expression system.
- If an inducible expression system is already being used, make sure that the expression is not leaky.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest.
Check both cellular lysate and the medium for the presence of the protein of interest. While most proteins will be secreted with a secretion signal sequence, it is not guaranteed for all proteins.
The protein of interest may be lacking post-translational modifications that are required for functional activity. For example, chorionic gonadotropin requires mammalian glycosylation to be active. While insect cells do provide glycosylation, they do not provide the specific glycosylation required for chorionic gonadotropin activity.
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