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phosphorylation analysis - (Mar/20/2006 )

Greetings to everybody,

i have a little problem and i want your precious help.
after incubation for 12 hours of mammalian cells with a certain peptide, i observed that now they grow more rapidly. so i want to check if the presence of the peptide triggers a cascade inside the cell and proteins get phosphorylated and if the expression levels of some proteins increased.
so i thought to run 2D DIGE gels before and after the presence of the peptide to see if some proteins are expressed more. the quantitation will be done using the melanie bioinformatics program. to check if some proteins are newly phosphorylated in the presence of the peptide i would run an autoradiography treating the cells with 32?P while growing...
1. any other ideas? what more controls should i use except the culture of the cells in the absence of the peptide?
2. can somehow calculate the cell number that i need to make this expreriment? how many cells to i need in number for each expreriment, for instance i am going to use total protein extract to run the 2D many cells do i have to use? if it depends on the amount of the protein, then how much is the lowest detectable amount on a 2D gel ?
3. if i want to check also the same question about the proteins inside the nuclear. any good protocols on how to isolate nuclears and then the proteins inside? cell number that i have to use, so to detect protein in 2D gel??..

Thank you very much in advance for any help!!!!


for question 2 i can't reply as i don't know the answer...
For question 3, i've posted a protocol that goes well in my hands. See here

for question 1: i though that maybe you an see if major pathways proteins are phosphorylated. If you have a clue of which one to see, phospho specific antibodies may do the job.
Mass spectrometry can tell you if a protein is phosphorylated too. But i don't think it's recommended to a global analysis... sad.gif


thank you very much! your answer is highly appreciated. this protocol looks promising.

i still search the number of the cells..i think 10 to the seventh should be ok...
protein expression levels probably will be checked with DIGE 2D electrophoresis.