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Low level of a Flag fusion protein was detected in Western. Why?


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#1 Licatchlove

Licatchlove

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Posted 26 January 2011 - 04:19 PM

Hi, I have problem with detecting a protein in Western blot. The protein is a fusion protein of interest protein with Flag, and it was inserted in a PcDNA vector with T7promoter. I transfected 293t cells. I searched on the internet, found a post had exactly the same problem with mine. But there wasn't any chase-up afterwards. The webpage is http://www.protocol-...sts/22226.html. I copy the question here.

Thanks in advance for any help and advice and for reading this long question!

The plasmid encodes a secreted protein and should be detected in both cell lysate and media. The protein is FLAG tagged and the vector is pCMVtag. A previous postdoc got very high expression of the protein in both cell lysate and media as expected, however I cannot achieve this result using the exact same protocol.

I get very low expression in cell lysate and very low or nothing in media.

However the strange thing is using another plasmid (also FLAG tagged, in pcDNA3 vector) enocding a different secreted protein I can get high expression and see the protein in both cell lysate and media using all different transfection conditions.

I cloned the insert from the plasmid with low expression into the pcDNA3 plasmid and it did not improve the results.

I want to use 293T cells, and I also tried 293. I used low passage number cells and I tried different stocks of frozen cells.

I tried both Fugene 6 and Fugene HD. For fugene 6 I tried a 1:6 or 1:3 DNA:Fugene ratio. For Fugene HD I tried 2:3 and 1:8. I used a ug amount of DNA that was in the suggested range (for Fugene 6 it was 0.4 ug in a 24 well plate). I also tried using 1 ug of DNA.

I followed the Fugene protocols exactly.

I used various confluencys as suggested by the transfection reagent (~50%, 70%, 80%, and 90%). I also counted cells and used the amount suggested (I tried between 50,000- 200,000 for both transfection reagents).

I did not get a lot of cell death and the cells are negative for mycoplasma.

I cultured the cells between 2-5 days after transfection (changed to serum free media 1 day after transfection). The previous postdoc got high levels of protein expression on day 2.

I tried using 1% serum in OptiMEM instead of serum free for the 2-5 day culture. Otherwise the media is always DMEM/F12 (10% FBS) for the transfection and I tried with or without antibiotics (but always without antibiotics and serum for making the fugene/DNA complexes)

For the plasmid with high expression and the one with low expression I purified them the same way using Qiagen midi prep kit. I also tried using stocks of the plasmid purifed by someone else with no difference in results. A260/280 ratio was 1.8. I ran the DNA on a gel and it was not degraded. The DNA is in TE at a concentration of 0.5 ug/ul. Phenol/Chl extraction and EtOH precipitation after the midi prep did not affect the results for either plasmid.

Sorry this is REALLY long! I greatly appreciate any help and advice, I am out of ideas!!
Zona Pellucida

Edited by Licatchlove, 26 January 2011 - 04:26 PM.





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