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Home Support FAQs Over-expression Cell Lysate FAQs

Over-expression Cell Lysate FAQs

1. Q: What makes OriGene the optimal source for over-expression cell lysates?
A: All of OriGene’s over-expression cell lysates are prepared from HEK293T cells transfected with TrueORF cDNA clones. Each expressed protein has a C-terminal Myc/DDK tag which can be monitored by anti-tag antibodies for expression verification. OriGene has the most comprehensive genome coverage in terms of ORF clones.
2. Q: What makes OriGene the optimal source for over-expression cell lysates?
A: All of OriGene’s over-expression cell lysates are prepared from HEK293T cells transfected with TrueORF cDNA clones. Each expressed protein has a C-terminal Myc/DDK tag which can be monitored by anti-tag antibodies for expression verification. OriGene has the most comprehensive genome coverage in terms of ORF clones.
3. Q: What are the terms of OriGeneGÇÖs over-expression cell lysate guarantee?
A: We guarantee that our over-expression cell lysates pass through stringent Western blot test using anti-tag antibodies. Each lysate is supplied with Western blot image showing positive protein band at or near the correct molecular weight. No guarantee is provided for other assays beyond Western blots.
4. Q: How long can I store these lysates?
A: OriGene guarantees 12 month stability from date of shipment when these samples are stored at -20 degree C. Avoid repeated freeze-thaw cycles. In our experience with some lysates, up to 5 freeze-thaw cycles in RIPA buffer does not cause significant protein degradation.
5. Q: Does your SDS buffer already containing reducing reagents?
A: Yes, the SDS Sample Buffer already contains 2.5% b-mercaptoethanol. There is no need add additional chemicals.
6. Q: Do all TrueORF clones produce same amount of over-expressed proteins?
A: Each clone is different. It is difficult to predict or control specific protein expression level. We provide over-expression cell lysates as 100 ug total protein with Western blot proof.
7. Q: Can I use these over-expression cell lysates for other applications beside Western blots?
A: The antigen standard is supplied in liquid format in RIPA buffer. One can use our standard for immunoprecipitation, ELISA standard, protein/protein interaction, etc.
8. Q: How many Western blots can one do with 100 ug total protein in a lysate?
A: 100 -¦g lysate is sufficient for 20~50 reactions. Depending on over-expression level and antibody affinity, some lysates can go as low as 0.1-¦g per load.
9. Q: Does OriGene sell protein specific antibodies?
A: Yes, OriGene does sell some antibodies which have been validated in Western blot applications. Please check our website..
10. Q: What do I do if I can't find specific Lysate of my interest?
A: OriGene is routinely adding new Lysate products on a quarterly basis. Please check our website frequently or fill out the following feedback form to help us prioritize new product release schedule. OriGene will contact you as soon as the Lysate product you requested is available.
11. Q: Does OriGene sell antibodies for Western blot applications?
A: Yes, in addition to gene specific antibodies, OriGene also offers HRP labeled goat-anti-mouse IgG and chemiluminescent reagents for Western blot applications.
12. Q: Does OriGene sell Western blot MW standard?
A: Yes. We have a MYC/DDK Tagged Western Blot Molecular Weight Markers (MWM1001) which can be used in WB applications.
13. Q: What source do you use for the predicted molecular weight of the antigen?
A: We mainly use the swissprot webpage to help predict the molecular weight of unprocessed protein. Please see the following link: ExPASy Link
14. Q: If the western-blot analysis shows that the immunoreactive band is different from your predicted molecular weight, does it mean that I did not get the right over-expression lysate for my gene?
A: The predicted the molecular weight is calculated from the protein primary amino acid composition. There could be some discrepancy from the actual molecular weight, if there were post-translational modifications or known proteolytic activity. In this case, OriGene strongly suggests researchers check related literatures on this particular protein.
15. Q: If the SDS samples have been frozen, do I have to re-boil them before loading?
A: Yes, it is recommended. It will help to denature the protein better.
16. Q: I am working on a multi-span transmembrane protein. Should I boil my sample before SDS-PAGE fractionation?
A: No. According to our experience, the boiling of transmembrane protein in SDS sample buffer tends to induce protein aggregation. We suggest that the customer incubate the over-expression lysates with 1xSDS sample buffer at room temperature for 30 minutes before loading.
17. Q: Why sometimes I see more than one bands with some lysates in Western blot experiments?
A: This happens from time to time because of post-translation modifications, protein degradation, etc.
18. Q: I need to cite your product for a paper I am writing. What language should I use?
A: We recommend that you refer to the product by its specific catalog number and refer to us as OriGene Technologies (Rockville, MD). Furthermore, we'd love to hear from you when your paper is published. Inform us and we will send a gift.

 

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