How many kDa add a post-translation modification? - (Nov/06/2010 )
Hi,I am a new member of this forum. I am sorry in advance for my english and if there is a similar topic. I tried to look it but I would have to know... how many kDa can to add each post-translation modifications? For istance Phosphorylation, Ubiquitination, Acetilation, etc...
My target protein once activated translocates into nuclear enviroment, in this way can to regulate trascrptional event.
From literature I founded a lot of phospho and acetilation and 2 Ub sites.
In my Cytosolo Nuclear exctrats I find a new band that it distances 8-10 kDa about from datasheet reported band. Expected band get out normally but in presence of newly band shows less intensity.
I find it in Nuclear extracts alone, both in Primary culture and Cell Line samples.
Thanks
Unsurprisingly the size difference between PTM and normal bands is dependent on the number of modifiations. Phosphorylation is the smallest and usually shows as a smear rather than a discrete banding, as does acetylation. Sumo is about 12 kDa, and Ubiquitiniation is about 8.5 kDa/Ub if I recall correctly, so it is quite likely you have a mono-ubiquitinated product.
You should check that the larger band you are seeing is specific. If you know the epitope of the antibody you are using you can pre-incubating the antibody with a peptide that has the same sequence as the epitope, then using it to probe the western, which should block binding. The other way to do this would be to knock down the protein of interest and demonstrate a loss of both bands.
bob1 on Mon Nov 8 00:16:00 2010 said:
You should check that the larger band you are seeing is specific. If you know the epitope of the antibody you are using you can pre-incubating the antibody with a peptide that has the same sequence as the epitope, then using it to probe the western, which should block binding. The other way to do this would be to knock down the protein of interest and demonstrate a loss of both bands.
Thank you very much to answer me promtly. Two ideas you suggested me should confirm ab specificity to larger band. Then, I would be positevely able to hypothesize a PTM. However, the monoUbq-status could be interesting because disappears progressively when protein of interest is not required.