Hello everyone,
I am using lentiviruses to overexpress proteins (Myc-tagged versions) of interest in hepatoma cells. After several rounds of selection with G418, all the untransduced cells were dead, and the transduced ones had expanded.
I took samples to detect the protein by Western blot. I have an antibody specific for this protein (Ago2), that I know works well. Additionally, I also performed a Western blot against the Myc tag.
As a positive control, I used a sample of cells that I had transfected with a plasmid that expresses the same Myc-tagged version of Ago2 (N-terminal tag).
To my surprise, I could not detect specific bands using the specific anti-Ago2 antibody in the cells transduced with the lentivirus but I did see it in the cells transfected with the plasmid. The anti-Myc antibody showed specific bands in both lentivirus and plasmid transfected cells.
Note: These cells are Ago2 KO cells that I made with the Crispr/Cas9 technology, using plasmids to transiently express both Cas9 and the guide RNA. Now I need to get Ago2 back into these cells to perform complementation assays.
Any ideas of what might be going on here? Is it possible that only the Myc tag is expressed and not Ago2? Is it possible that Ago2 is being degraded for some reason?
All ideas will be very much appreciated!
Hi Jenna,
Thank you very much for your reply.
I don´t think that that is the case because I have a plasmid with the same tagged variant (I digested the plasmid expression vector to take out the band and cloned it in the lentivirus expression plasmid). It is being expressed by the plasmid and it is recognized by the antibody. A sample from cells transfected with such plasmid was what I used as a positive control and I got bands both with the specific antibody and the anti-Myc.
Hello Yalena,
The suggest from Jenna is correct from my perspective because no one (to my knowledge) is able to predict the quarternary structure of a protein or even can exclude that adding a single amino acid or even a full peptide or protein has no influence on your protein tagged with a full myc.
Your example with another protein which was tagged with the myc and detected by the specific antibody is not applicable universally to all proteins!
As you have written above that you add the myc on the 5´ end of Ago2 it can be better to try a 3´ tag which is maybe interfering less with your protein of interest and its folding respectively.
If you want to avoid that new cloning procedure I would look for other myc specific antibodies and test them, maybe one is among them who is binding an accessible AA sequence in your 5´ myc tagged Ago2 and gives you a specific band. Review if a publication about myc existst with this antibody verifying its specificity.
Regards
Hello Volker,
Thank you very much for your reply.
I don't know if I have explained this clearly...
I have the same Ago2, tagged in the N-terminal with a Myc tag, both in the lentivirus vector, and in pcDNA3 vector. I made the Ago2 lentivirus vector by cloning the Myc-Ago2 band digested from the pcDNA3 vector into the lentivirus vector. Then I transduce some cells with the lentivirus and others with pcDNA3.
Result: I detect the Myc tag in both of them, but Ago2 only in the ones transfected with pcDNA3.
Is it possible that two different expression systems (lentivirus vs transient expression with a plasmid) give such different results in terms of protein folding and accesibility to the antibody by Western blot?
Additional information: I do denaturing SDS-PAGE before Western blot, so my guess is that the protein is completely denatured and the original folding shouldn't interfere.
Have you made a control digest of the lentiviral vector and sequenced your? Maybe you have cloned in a turncated Ago2? Then the part which is recognized by the antibody is maybe missing or a nonsense transcript is created.
When you use a denaturated SDS-PAGE then is should be anyway accessible, you are right!
Hi Volker,
I sequenced the vector after cloning and couldn't detect any problems. One more thing that is intriguing and not consistent with the idea of a truncated variant being expressed, is that the band detected with the anti-Myc antibody shows the size expected for a full lenght protein. Now I am thinking that maybe it is just that I have very low levels of expression, such that the anti Ago2 cannot detect but the anti-Myc does...
- Badges
- Science method