protein band is shifted after incubation with plant protein extracts! - (Oct/24/2007 )
Hi all,
we are currently studying a transcription factor. We produce this TF fused to His tag in N-term and Strep-tag in C-term in E coli. When this protein is incubated with a plant protein extract and then purified thanks to the tags, we were able to observe 2 bands on gel (using silver staining and antibody against His-tag). One band is higher and the other lower than the tagged protein that has not been mixed with protein extract. Is it possible that the TF has been modified after incubation with the plant protein extract? And if so, how can i figure out the kind of modification that has occured? Can Mass spectometry help me?
thanks
we are currently studying a transcription factor. We produce this TF fused to His tag in N-term and Strep-tag in C-term in E coli. When this protein is incubated with a plant protein extract and then purified thanks to the tags, we were able to observe 2 bands on gel (using silver staining and antibody against His-tag). One band is higher and the other lower than the tagged protein that has not been mixed with protein extract. Is it possible that the TF has been modified after incubation with the plant protein extract? And if so, how can i figure out the kind of modification that has occured? Can Mass spectometry help me?
thanks
Hi,
I am not expert, but I would consider and ask (myself):
After incubation of your protein with plant extract there are two bands and the first one has higher molecular weight and the second one lower Mw in comparison to protein which was not incubated with that plant extract. So there is not any band with the same molecular weight (when compared plant extract incubated and non incubated protein)?
1. How big is the shift of molecular weight between those two bands that you can observe (or in comparison to non incubated protein)?
For example, the lower band can be some cleavage product (cleaved by some plant protease) and the upper band can be your protein interacting with other component (protein) in plant extract or if the Mw shift is very small, then it can be more likely some modification of your protein.
2. Do you incubate pure protein with the extract or some cell lysate?
2. What contains your buffers? Are there any protease inhibitors, phosphorylation or dephosphorylation inhibitors, reduction agens? What are the conditions of incubation with that plant extract? Is it whole plant extract or only selective part of plant?
You can try to use different inhibitors of proteases, glycosylation, phosphorylation and you will see what happens.
You can try to do in vitro deglycosylation. You can try to do 2-D gel to get some hint about modification of your protein (phosphorylation gives more negative charge to proteins). MS can also find some modifications of protein (phosphorylation, glycosylation and other), especially if you have high amount of pure protein it is very meaningful (and possibly helpful) to try MS.
Thanks for sharing your thoughts. I think also that MS would help but i don't know how...I have to tink about it as i have no experience on it