I had cloned my gene of interest in to a vector, which has fluorescent protein tag. I did restriction digestion and did insert release of 1.4kb and fragment release having both gene of interest and GFP tag at 2kb. I did transfection in to N2a cells and expressed protein and did western blotting. I was supposed to get a single band at 85kd. But, I also got a band at 60kd, which is also detectable by both antibodies specific for the gene and gfp. what could be the reason, for getting the lower band at 60kd, which is also my gene along with the tag. while I got everything done right in colony pcr and clone confirmation. How to overcome this problem? Kindly, help me.
First lane is my test sample. 2 bands at 85kd and 60kd. second lane is my positive control and third is my negative control.
Dear Megha,
In all probability it appears to me that, either may be the cDNA which you have inserted/cloned, contains two ORF with two distinct/separate transcription start sites (Check for it using NEB Cutter!) or the protein product from the cDNA is expressing two different but very closely identical splice variants. In both the cases, both products is carrying the specific epitopes for the antibody, hence are getting detected.
And as far as I assume, your GFP must be C-terminal, which is why it is being always retained to express and is getting detected too.
From your data above it also appears that the lower variant is expressing in a much less amount compare to the higher one, hence to me, would not be much of a problem.
But still I will suggest you to carry out a thorough literature search in this regard to get ensure.
If you are desperate to clear out the lower band then, you must try for a different antibody which has been prepared from a different region of epitope from the one you are currently using, but before doing this, you need to do a protein-blast to confirm the exclusiveness of the epitope between the variants.
Good luck!
Hi,
I agree with Anirban. To me it also seems like it is a truncated product of your fusion protein, with presence of epitopes for both the antibodies. Try to find out if there are any ribosome binding sites and/or internal transcription start sites in your GOI.
From your description, I can tell that your cloning is clean. I would suggest you to check your sequence of the gene.
Also, check out if any protease site is present around that region (~60kDa= Around 545-550th amino acid residue=somewhere near 1600-1650th nucleotide position)
Hope this helps. Please keep us updated so that we can help/learn from your experience.
Good luck... :)
Hi, thanks for your suggestions.
Dear Anirbhan,
I subjected my sequence for ORF finder and found the below result.
You mentioned about protein blast of 2 varients.
can you please help me in this regard.
Thanks!
Hi Shridhar,
I checked for start site in GOI at around 564bp and protease cleavage site present at every 10 amino acids.
If truncation of protein has taken place, when could it happen?
What is the solution?
Should, I repeat the cloning again?
One of the literature quotes the below reasons for truncation.
• Interrupted translation, which leads to a variety of truncated protein products.
• Frame shifting.
• Misincorporation of amino acids. For instance, lysine for arginine as a result of the AGA codon. This can be detected by mass spectroscopy since it causes a decrease in the molecular mass of the protein of 28 Da.
• Inhibition of protein synthesis and cell growth.
Hi Megha,
I don't think you need to repeat the cloning as there is a successful expression of the GOI, the aim is to minimize/remove the unwanted band.
The problem of truncation either lies at handling level or at molecular level. Let's not jump to the molecular level as of now. If it is possible, tell me your expression conditions and how do you process the cells post-expression.
Also, I had a quick thought, internal start site is not the problem. The real problem is internal transcription termination. I assume that fusion partner of your GOI is at N-terminal of the protein. I'll get back to you on this asap.
Many times it happens that solution is much simpler than we think. So I'd suggest you to check protein handling, if that's fine then we can always go back to repairing at molecular level.
Hi Shridhar,
Thanks for your suggestions. My GOI is at C-terminal.
well, one of my senior had repeated with transfection , protein expression and did western blotting. She too got the same results as mine. But, in her case, the GOI expression was more than mine and the lower band with minimal expression like mine. Seems like, their is no problem here. But, when i got my clone confirmed with sequencing or restriction digestion it was fine.
I have posted my GOI's ORF . Do you think, their is any other open reading frame or stop codon ?
I agree with Shridhar's suggestion. In addition you may try some protease inhibitor cocktail while you lyse your cells that may stop undesirable truncation in the protein. On another note, is it possible that your fusion protein could be a dimer? Run a non-denaturing condition of SDS-gel and then do the Western blot to figure out that possibility.
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