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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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hard-to-detect proteins in mammalian cells
Posted by: ivylee02 (IP Hidden, New member, 3)
Date: July 27, 2005 11:20PM
Hi,
I've tried to overexpress 4 different HA-tagged proteins in 2 different cell lines (HEK293 and K562) with 2 different vectors, one with an IRES-GFP and one with geneticin-resistance. I've tried Western blots with ECL/ECL plus detection and I don't really see my proteins on these blots. For K562, I did not see any proteins on the blot transient or stable. For HEK293, only transient proteins were detected and not stable. however, when I did some functional assay with the stable K562 cells, the proteins (although undetected) seemed to give some activity so that might indicate that the proteins are there but just couldn't be detected... Also, for the stable 293 cells, under immunofluorescence microscopy, I looked at a few cells and I saw that a small amount of the cells have the protein but just couldn't be detected on a Western blot... So I'm thinking that either all 4 of my proteins are unstable long term or that I need a better way of concentrating the protein-expressing cells. (I'm trying IP now and see if I see something tomorrow.) Does anyone have similar experience or any idea about this? Thanks!
Re: hard-to-detect proteins in mammalian cells
Posted by: hayes (IP Hidden, Unregistered user, )
Date: July 29, 2005 11:20AM
hi ivylee, you only painted partial picture here which makes troubleshooting difficult. Your problem itself looks like a GRE logical exam problem. OK, so there are 4 genes cloned into 2 vectors, GFP and G418 resistant vectors. There should be a maximum 8 final constructs. If all of them were transfected into 2 cell lines there should be maximum 16 transfected cultures. I am a little confused but let's continue but stop the math calculation. Protein detection by western shows nothing for K562 and stabled transfected 293. Only transient transfected 293 has at least one of your HA-tagged protein expression (or all of the four proteins?). Protein detection by IF staining shows suspected expression of at least one of your HA-tagged protein (or all of the four proteins?), which is somewhat contradictory with your western result.
My answer: 1. The antibody may not be able to detect your protein. I also experienced antibody against tag (Flag not HA though) from some vendors are not good at all. 2. The stable cell line should express your protein unless you selected them in a much less straingent condition and caused mixture of wild type cells and transfected cells. I could be wrong and score zero :)
Re: hard-to-detect proteins in mammalian cells
Posted by: ivylee02 (IP Hidden, New member, 3)
Date: July 29, 2005 08:44PM
Hello hayes, you are right so far on the background story. So for the transient 293 (not stable 293), I see all of my HA-tagged proteins. Transient and stable K562 don't show any expression on Westerns. Protein detection by IF staining for STABLE 293 cells (I only had time to look at a few cells for one protein) showed some proteins. I don't know if I'm having a stability issue with all 4 of my proteins because the selected cells (based on GFP or geneticin-resistance) somehow seem to have "lost" a lot of the proteins.
My real-time RT PCR results show that the transcript levels are good. The antibody I used was able to detect the proteins in transient 293 (and also in vitro translation). Although proteins undetected, the K562 "overexpressers" do show some activity compared to the wild-type cells. Any ideas are welcome! Thanks!
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