unexpected western blot band - western blot (Jul/27/2008 )
I used 15 amino acids of a cucumber DNase protein as antigen to produce antibody in rabbit. I use this antibody and get good result of the protein expressed in Ecoli.
The active DNase content in total protein is very low. I can detect it by in gel activity assay, which is very sensitive. The DNase activity increase during the leaves grow. When I do western blot with the protein extracted from cucumber leaves, there is only a very stong band about 7kd bigger than expected(37kd). I can not find the right size band.
One possibility is the antibody react with other protein. The other possibility is the bigger band is a modified DNase.
How can I test the two possibility?
Thank you very much for your comments!
-seetide-
QUOTE (seetide @ Jul 27 2008, 05:40 PM)
I used 15 amino acids of a cucumber DNase protein as antigen to produce antibody in rabbit. I use this antibody and get good result of the protein expressed in Ecoli.
The active DNase content in total protein is very low. I can detect it by in gel activity assay, which is very sensitive. The DNase activity increase during the leaves grow. When I do western blot with the protein extracted from cucumber leaves, there is only a very stong band about 7kd bigger than expected(37kd). I can not find the right size band.
One possibility is the antibody react with other protein. The other possibility is the bigger band is a modified DNase.
How can I test the two possibility?
Thank you very much for your comments!
The active DNase content in total protein is very low. I can detect it by in gel activity assay, which is very sensitive. The DNase activity increase during the leaves grow. When I do western blot with the protein extracted from cucumber leaves, there is only a very stong band about 7kd bigger than expected(37kd). I can not find the right size band.
One possibility is the antibody react with other protein. The other possibility is the bigger band is a modified DNase.
How can I test the two possibility?
Thank you very much for your comments!
I dont know how you can test but I just want to know is it possible your protein become dimerized?
-saraarasus-