Western Blot with transient transfected cells

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schrl
schrl's picture
Western Blot with transient transfected cells

I can't detect GPCRs in transient transfected cells via Western Blot. Cell lysates were harvested 24h after transfection. I get a really strong band with a myc antibody which detects the tagged protein and I get bands in Western Blots with tissue lysates. I recognized this problem with several GPCRs now. Do I have to conclude that my antibody does not detect the expected protein? Does anybody has an idea or suggestion?

saswati1
saswati1's picture
Please check first with a

Please check first with a positive control along with your samples. Sometimes, antibodies are not as good as you can expect. If I were you, I would first run a positive control to make sure that positive control works with that particular antibody before making any conclusion

Jon Moulton
Jon Moulton's picture
Might your myc tag change the

Might your myc tag change the conformation of the antigenic site where your GPCR antibody would otherwise bind? Running a comparison of the native GPCR and the myc-tagged GPCR at equimolar concentration would determine whether the tag is decreasing affinity of the anti-GPCR antibody.

schrl
schrl's picture
Thank you all for the

Thank you all for the information! What I am wondering is what to use for positive control. Is there a better way than to use transfected cells vs untransfected cells? I also loaded tissue samples with high expression level but who can tell me if I get a specific signal in here? The conformation is a good point. I also thought about that and transfected cells with untagged, C-terminal-myc and N-terminal-myc and did not get any bands in all cells although I get signal in tissue samples. My signal in tissue samples maches with the expression level results but without a clear positive control I can't be sure to detect my expected protein. :-( What do you think?

kumar
kumar's picture
Hi Schrl, your strong band

Hi Schrl, your strong band with myc-antibody tells me that the GPCR is being expressed. Your results seem to imply that the GPCR antibody you are using does not work in transfected cells. That raises the questions: Was it raised against a synthetic peptide? or Do the transfected GPCR undergo same post-translational modifications as in the tissues? You can prove that antibody is not working by showing a lack of receptor blocking activity in transfected cells compared to in the tissues.

schrl
schrl's picture
Hi Kumar, the post

Hi Kumar, the post-translational modifications are a good point. Maybe I should try another cell line? How would you prove the lack of receptor blocking activity? What I already know is that my cells work well in reporter assays.

The antibody is raised against a synthetic peptide like most of the GPCR antibodies I know. By the way: does anybody know a supplier, which offers GPCR antibodies against the whole protein?

kumar
kumar's picture
You can show the lack of

You can show the lack of receptor blocking acitivity by assessing the Calcium, IP, or cAMP responses etc. depending on which receptor type you are working with. If the antibody works then the receptor activity can be expected to be blocked, if not then receptor should still signal.

http://www.nature.com/bjp/journal/v141/n1/full/0705592a.html

I have come across companies like Multispan (U.S.A.), Genovac (Germany) and M-phasys (Germany), that generate antibodies against native GPCRs.

schrl
schrl's picture
Hi Kumar, thank you very much

Hi Kumar, thank you very much for your helpful answer. The blocking assay seems to be a really good assay for checking the antibody. Thank you also for the companies. That sounds also very interesting.