Our truncated protein expressions (labeled by the HA tag) in lane 4 and 5 were supposed to be matched with the mRNA expressions in lane of 293FT-4 and 293FT-5. However, the different truncated proteins seems to be equal of molecular weight. We changed different vendors of HA antibody but the results were the same. And we are sure our truncated protein expression sequences are correctly with Kozak sequence followed by the initiation codon and stop condon, and the CDS length is the integer folds of 3, thus it seems to be an error of protein translation.
Any suggestions? Thanks a lot~!
Could be contamination with your WT plasmid (non truncated)?
From the PCR figure, it is not clear how big a difference really is in your truncation.
A difference of only a few amino acids will not be seen with a 90 plus kD protein.
Hi there,
I'm puzzled about the sizes of mRNAs (btw I suppose the figure is showing PCR products, not mRNA...) and proteins: they don't fit. 1kb is possibly encoding a 30kDa protein but you are showing mRNAs (PCR products?) which are less than 1kb and corresponding proteins which are far more than 30kDa... What is the exact protein size shift you are expecting?
Dear Amit V Pandey, thanks for your reply. We have checked the potential contamination of plasmid DNA by PCR without RT, and showed no bands existed. So we are sure there is no contamination by the plasmid DNA. The designed truncation lengths of proteins are 955bp and 559bp, thus the difference is 396bp long.
Dear Dominique Liger, thanks for your reply. The total length of CDS region is 2721bp, the truncated length is 2721bp-396bp=2325bp. we designed a pair of primers which could amplify the middle CDS region (955bp) that contained the truncated sequence (396bp).
An additional question is, our overexpressed CDS region didn't followed by the poly(A) signal, does that really matter to translation? Could this explain the unusual phenomenon?
Hi again,
If the host for expression is eucaryotic then polyA is definitely essential... About the WB: did you run a negative control sample issued from a cell transfected with an empty plasmid? It is just to make sure the band is specific to the presence of your gene...
Dear Dominique Liger
Yes, the lane 1 and 2 were the cells transfected with empty plasmid with FLAG tag, but incubated with HA antibody, thus the bands appeared to be not specific.
However, I think we are close to the answer of why our protein results being abnormal, it's highly possible due to the loss of polyA sequence after our CDS sequence, and the expression vector was not a lentiviral vector, so CDS must followed by a polyA sequence.
So the truth should be the bands were unspecific staining and the wanted proteins were not successfully translated.
Sincerely Thanks!
I suggest using standard cloning sites in vectors that include your tags. After pcr amplification to include the cloning sites in your fragments, digest both the vector and your fragments with enzymes, and ligate using standard protocols.
You may need to remove stop codons if a c-terminus FLAg/HA tag is needed. In that case, vector will have a stop codon after the tag sequence.
I am suspecting something is misaligned in your final clone and no protein production is happening.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression