If you think GFP is to big you could use the splitted vertion of GFP (the one that is usually used for Bimolecular fluorescenc complementation experiments). I am possitive that anti GFP antibodies recognize at least one of the two halves of GFP protein. The Mw is around 12-15 KDa. Another option might be trying to clivate your protein with a protease or a chemical method. If you can clivate your protein just in a few sites you might be able to pay atention to the N-terminal fragment and see the difference. Another option might be adding an speciffic protease sensitive secuence inside your protein f you have idea were you could do that without altering its folding.

May be very naive, but why not insert a stop codon after approximately 10 kDa (100 aa, thus 300 bp) in your big protein

@Patrick. Thanks you for your suggestion. However, I tried it already but the protein was degraded although I have done careful analysis of protein's domain organization.

@Aaron. Thank you for your idea. I have already the constructs where I have inserted 3xHA-3xFLAG cassette after 15th and 45th codon. Unfortunately, both of them did not give any signal on western blotting.

Or if you know the structure of your protein, insert a stop after the first folded domain

Maybe you can amplified by PCR your 10-15 codon with degenerated oligonucleotides to add restriction site and to cloned this fragment in a vector with GFP fusion tags in Cter. GFP is around 27 KDa, is inert and many Antibodies are efficient.

Aaron Irving uis i think right can you add gst tagg

Maybe it could be useful to generate monoclonal antibodies that recognize only the 8 amino acids; you have to inject your protein into an animal with coadjuvant; select the B cells that produce antibodies against your protein and hybridize with a myeloma cell.

Then you have to prove all the antibodies produced and detect the one that recognize your 8 amino acid plus protein and does not recognize your normal protein; a large procedure but may have good results

@Nicolas and Prashant. GST and GFP are too big in my opinion. I have to detect the difference in 0.9 kDa. Or which percentage of AA in my SDS-PAGE do I hav eto use to distinguish between 27 and 28 kDa?

@Manuel. In general, it is a good idea to develop Abs. Unfortunately, I am restricted in time and money. And the sequence of my additional 8 amino acids is MRARRTVL which I did not specify above. Aren't R-rich peptide are lucrative substrates for proteases?

You could insert a phosphorilable sequence which is very very small and inert but can be detected with high sensitivy with radio labelled substrates

@Marco. It's great idea! But since I am working in E. coli, I probably should go for a sequence which can be in vivo biotinylated.

If you think GFP is to big you could use the splitted vertion of GFP (the one that is usually used for Bimolecular fluorescenc complementation experiments). I am possitive that anti GFP antibodies recognize at least one of the two halves of GFP protein. The Mw is around 12-15 KDa. Another option might be trying to clivate your protein with a protease or a chemical method. If you can clivate your protein just in a few sites you might be able to pay atention to the N-terminal fragment and see the difference. Another option might be adding an speciffic protease sensitive secuence inside your protein f you have idea were you could do that without altering its folding.

@Martiniaro, @Bastian. Thank you very much! Your suggestions are very useful.

here you can find some guys that used anti-GFP antibodies to detect the N-terminal of the splitted YFP

http://www.plantphysiol.org/content/145/4/1090.full

You may protein blast your peptide MRARRTVL restricting search for human or mouse only and you will get proteins that have similar stretch of amino-acids. than search for mAb that bind the protein found and check is epitope sequence known if you lucky you might get a hit. if not go for polyclonals for that protein and do a low stringent western blot. Need lots of controls but possible. I did similar thing, it works.

Ubiquitn mw=8656 Da) would serve as a very good candidate. It is stable and appending of an additional peptide to the N terminus is known not to destabilize the protein. In addition, there are very good antibodies available for monitoring expression by Western. I suggest that you express without the C-terminal glycine dipeptide so it is not subsequently conjugated.

You could try cruciferin or some other seed storage protein. Cruciferin is 12kDa and quite inert, however you might get aggregations forming in the cell.

Alternatively, have you considered fractionating your protein with a protease and separating the fragments by mass spectrometry? That would be very fast and you would know exactly what peptides you get.

I am of the similar opinion as made by Martiniano Ricardi. It should work. Please try this time and find out whether it works , and let us know. Thanks

What about running SDS-PAGE cutting out band where you expect your proteins and sending the gel slab to LC-MS-MS/MS for identification

@Marcin and Lester. MS will be considered when I find optimal condition for my protein to be translated in vivo. I mean type of stress and exposure (initial phase, recovery phase, etc.). Before that I need a fast and robust method for detecting my protein, at least as fast as WB.

You can also fuse your protein of interest with repeats of well-known epitopes, for example triple HA epitope can be easily detected by western-blotting. The more repeats you use the more sensitive detection of your protein will be, so you can use less material for analysis. 3xHA can be easily cloned upstream of the gene by insertion of doublestranded oligonucleotide (it is about 87 bp).

Another option is to contact the VIB laboratory in Brussels for a good expressing Nanobody (15kDa) as reporter protein