I'm going to transiently transfect primary pre-adipocytes with His-tagged plasmid DNA [pcDNA3.1(+)] containing ADIPOQ gene with a SNP to study the gene and protein expression by using FuGENE 4K transfection reagent.
I would like to ask what is the most appropriate protocol to use in order to determine the successful rate of the mentioned transfection?
I was planning to use Pro-DetectTM Rapid His Competitive Assay Kit from ThermoFisher (A38507) for transfection confirmation. However, I am wondering how do I select the best transfection dilution that give the best transfection rate using the competitive assay kit.
Is it possible for me to solely rely on the number of lines appearing on the strip?
(As the concentration increases, the number of test lines will decrease until all test lines disappear. The concentration of the His-tagged proteins is inversely related to the number of test lines appearing on the strip).
Detail of the competitive assay kit is provided in the attachment, and this is the product link:
https://www.thermofisher.com/order/catalog/product/A38508?ef_id=CjwKCAiAmZGrBhAnEiwAo9qHia_rdYYp1DiRAC8VMtmH3mj0MsVJCVwy2qfr7Q5teJ67iIw-otp2SRoCLfgQAvD_BwE:G:s&s_kwcid=AL!3652!3!384464758933!!!g!!!6538554939!82560538550&cid=bid_pca_wwr_r01_co_cp1359_pjt0000_bid00000_0se_gaw_dy_pur_con&gad_source=1&gclid=CjwKCAiAmZGrBhAnEiwAo9qHia_rdYYp1DiRAC8VMtmH3mj0MsVJCVwy2qfr7Q5teJ67iIw-otp2SRoCLfgQAvD_BwE.
Million thanks in advance for suggestions and generous support.
I would recommend a Western Blot analysis first in order to determine the concentration of the His-tagged protein in your lysate. Use a His-tagged protein with known concentration (if availble) to determine (roughly) your expression level.
These lateral flow tests are less sensitive than Western Blots and if you are using a cell line that is not normally used for protein production, the yields might be too low to get a good signal. Especially, if you are trying to optimize transfection conditions where differences of e.g. 20-50% more in one condition could hardly be detected.
I have produced and used similar His-tag lateral flow tests in the past and compared them to the ones from Thermo Scientific. Small differences are not easy to detect using this principle plus it is protein dependent (sometimes the tag is not well accessible and the result might be misleading). Western Blot is not exactly quantitative either but definitely more sensitive and not so much dependent on the structure of your protein.
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