Hi, we recently transfected mouse ES cells by electroporation a construct containing Neo resistant gene and MT-I promoter driving expression a fusion protein with FLAG and 6X His tags. Positive clones selected were verified with 5’ and 3’ end pcr primer sets. Western showed a FLAG protein 130kd which is larger than expected 80 kd, and it cannot be recognized by target protein-specific antibody. However the positive clone has correct sequence after careful genomic sequencing, with no frameshifts or loss of stop codon being found. I am totally confused. How come the positive clone has correct integrated sequence, while it expressed wrong protein? Any thoughts and feedbacks are welcomed and appreciated!
1. Do you see this band in untransfected cells? This will make sure that the band you see at 130k is due to transfected gene.
2. If you don't see it in untransfected cells and you had verified the sequence, frames, mutations etc by sequencing, it could be post-translational modification
3. Make sure your stop codon is ok. Otherwise it might go on translation until next stop codon comes and might lead to 130kDa. When your protein specific antibody could not recognize the band, it is more likely frame shift change.
4. If sequence is not the issue there can be two possibilities.
A. Dimerization (an 160k might look like 130k in high % gels)
B. Glycosylation.
5. If it is glycosylation, it can be N-linked or O-linked glycosylation. O-linked glycosylations are usually short chains and hence does not shift a protein from 80k to 130k so it is likely N-linked glycosylation. To check that, treat transfected lysates with PNGase and then check whether the size is shifted back to 80k from 130k.
These are all just possibilities.
Hi,
I suppose the calculated mol w of your protein includes the additional 6His and FLAG, which however does not amount to 50 Kd.
If you checked your construct by PCR only, there is a possibility that your insert was introduced several times. It is not very frequent, but it can happen in some ciscumstances; One way to check this is by digesting your DNA with enzymes that do not cut inside the insert; An alternative explanation could be that there is an upstream ATG, and your 130 kd protein includes a part upstream; you can check this by looking for other ATG and STOP codons upstream of your insert.
Finally, your protein might be modified post-translationally: this may also explain the fact that specific anitbodies fail to recognize it.
Hope this helps!
1. Do you see this band in untransfected cells? This will make sure that the band you see at 130k is due to transfected gene.
2. If you don't see it in untransfected cells and you had verified the sequence, frames, mutations etc by sequencing, it could be post-translational modification
3. Make sure your stop codon is ok. Otherwise it might go on translation until next stop codon comes and might lead to 130kDa. When your protein specific antibody could not recognize the band, it is more likely frame shift change.
4. If sequence is not the issue there can be two possibilities.
A. Dimerization (an 160k might look like 130k in high % gels)
B. Glycosylation.
5. If it is glycosylation, it can be N-linked or O-linked glycosylation. O-linked glycosylations are usually short chains and hence does not shift a protein from 80k to 130k so it is likely N-linked glycosylation. To check that, treat transfected lysates with PNGase and then check whether the size is shifted back to 80k from 130k.
These are all just possibilities.
Thank you so much, Monica and Goodwin. Can N-linked glycosylation mask the epitope away from the modification site? The epitope is 396-495th aa, while predicted glycosylation sites are at 180, 321, 588, 597 and 680th aa.
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