Dear everyone,
I want to tagged GFP to my target protein. Do I have to insert a simple peptide such as GGGG between GFP and the protein? And how many amino acids is enough?
Thanks you so much.
Dear Long Duy Duong ,
Yah you can insert, but before inserting peptide, you should need to select biologically active peptide which should be required for your study.,
Simply you want to insert any peptide means not make sense know, i don't know whats your Aim.,
Generally 4amino acids minimum should be recommended for this condition, and you are just said GGGG., i thing GGGG is not god option, because it's 100%hydrophobic and not have any value, so you try peptide which is bilogically relevant to your study., such as VYGR like this peptide.
50% or more hydrophilic nature peptide was recommended and it means get bind perfectly, hydrophobic such as GGGG not recommended for your work.,
Hope you got, if any more doubt regarding to this feel free to get back to me,
Good Luck & Regards
R.Selva....
Dear Long Duy Duong ,
Yah you can insert, but before inserting peptide, you should need to select biologically active peptide which should be required for your study.,
Simply you want to insert any peptide means not make sense know, i don't know whats your Aim.,
Generally 4amino acids minimum should be recommended for this condition, and you are just said GGGG., i thing GGGG is not god option, because it's 100%hydrophobic and not have any value, so you try peptide which is bilogically relevant to your study., such as VYGR like this peptide.
50% or more hydrophilic nature peptide was recommended and it means get bind perfectly, hydrophobic such as GGGG not recommended for your work.,
Hope you got, if any more doubt regarding to this feel free to get back to me,
Good Luck & Regards
R.Selva....
minimum 4amino acid sequence maximum as for your wish, some hydrophilic peptide you find and insert. if GGGG means it wont be much active.,
Look at the C-terminus and N-terminus regions of the proteins that you are trying to fuse. If there is enough flexibility, four amino acid linker should be sufficient. I prefer to use GSGSS... type linkers. As mentioned above, GGGGG is hydrophobic and cause aggregation.
You may wish to use a few repeats of Ala-Gly-Ser. (I agree that GGGG is not a good option). Also, if you have space, you may want to insert a hemagglutinin (HA) or other epitope for future immunohistochemistry. Best wishes.
I really appreciate and thank you all for your recommendations. I will try with the combination of Ala, Gly, Ser. Another thing is that I want to insert GFP between the signal peptide and the coding sequence. My target protein supposed to be localized to the plasma membrane. I used some softwares to predict the signal peptide. I am going to insert GFP into right after the signal peptide. And I wondering does it affect the cleavage site and also affect the activity of signal pepdidase or not?
Best regards,
Dear Long Duy Duong.,
Ala,Gly,Ser, will give good effect, just maintain some hydrophilic condition that's all,
If you insert GFP between signal peptide/coding sequence means i hope your assumption is correct.,
So in this condition sure it won't affect the cleavage site but may be affect signal peptidase.,
Mai i know after this whats your plan? means final stage what you are expecting? if this is last stage of your work for this condition means possible to remote this problem, if further you need to process means some special plan you need to create.,
Happy New Year 2015
Good Luck & Regards
R.Selva.......
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