Dear Experts,
As I am doing proteomic profiling for particular fraction of cell.
By following the protocol I prepared my sample for LC-MS/MS analysis but the concentration of protein in that sample was too low so I was not able to quantify the protein then I thought to use nanodrop @280nm in order to equally load the protein of test and control but now I have the peak intensity list and in few cases there is no intensity in control while the there is a very good intensity in test. So, I am wondering, is there any to overcome this situation, I mean, can I use particular protein intensity for the normalization of test with control. I am stucked on this step. kindly guide me to carry forward the results.
Thanks in Advance.
Personally I wouldn't use a nanodrop to estimate protein concentrations, it gave me quite a bit of varying results in the past. I normally just do Bradford assays. What is the actual concentration you think you have?
And it may be that some peptides just weren't picked in 1 run over the other. Are you just comparing them in seperate runs or are they labeled?
Hello Amit,
As per my experience, BCA works best for protein concentration determination in such cases (i tried many methods, Bradford, nanodrop, qubit etc. but best method was BCA and every other method provided false positive r negative results). If you were not able to quantitate you can make a relative quantitation from peak intensities of control and test sample but to subject the same for proper statistical comparison you have to run your sample for 2-3 times so that you can get an SD and make a valid comparison. So, Yes you can make a relative quantitative comparison from peak intensity values but you need to repeat the experiment for confirmation of the same.
Hope it helps !
Dear Vincent and Ajay I am really thankful to you for your valuable suggestion.
But I would like to highlight, that, protein quantitation is the main issue and I can not run technical replicates because in one run all the sample is getting consumed.. yeah but I am running biological replicates so I will try to put Ajay's Idea in that case.
If you (vincent and Ajay ) have any better idea for the current scene then do let me know. Looking forward for your suggestions.
Thanks
Amit
Hello Amit,
I think, if you have availability of instrument in your lab and your sample is getting consumed in single run then you should run biological replicates. Other alternative is that you can pool the samples and then isolate protein and run it. By doing so, you will get a higher protein amount and will be able to run 2-3 runs in a single go.
All the best !
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