troubleshoot the protein expression - different protein size but the same construct (Aug/03/2008 )
Hi
I did my first successful cloning of my gene of interest( mutated). the wt gene is about 100kd and the expected size of the mutated gene is about 25. After the creating the construct i did transformation and picked the 7 several colony and screeen for my instert. All of the colony had the my insert (expected size) and then i sent for sequencing most of all had 99% homology to gene mrna from NCBI when blasted. Thereafter, i did a western blot to conform its expression but all all the protein had diff size ranging from 50, 37 , 25 and i dont know which one is the right one, i want to pick the colony that has 25 kd because is close to the expected size or ? i dont know others ways to confirm, one lab member told to check if ist inframe.. but i dont know what to do.
vickie
Hi Vickie,
I suggest you align the sequencing result with the reference sequence of your mutant to make rule out any base changes ( additions/ deletions that may alter the reading frame). Also make sure that your stop codon is intact.
All the best
Regards
Avinash
http://aviprem.gq.nu
I did my first successful cloning of my gene of interest( mutated). the wt gene is about 100kd and the expected size of the mutated gene is about 25. After the creating the construct i did transformation and picked the 7 several colony and screeen for my instert. All of the colony had the my insert (expected size) and then i sent for sequencing most of all had 99% homology to gene mrna from NCBI when blasted. Thereafter, i did a western blot to conform its expression but all all the protein had diff size ranging from 50, 37 , 25 and i dont know which one is the right one, i want to pick the colony that has 25 kd because is close to the expected size or ? i dont know others ways to confirm, one lab member told to check if ist inframe.. but i dont know what to do.
vickie