I have a general question regarding which method would you recommend me to use if I would like to investigate the difference in the level of several proteins in tissue samples and compare different treatment groups. I could either do an LCMS/MS or a Western blot. Which one would you recommend and would you do both for validation? I am looking for time- and cost-effective techniques. I'll appreciate any suggestions. Thanks!
Western blot, though is a commonly used technique and is relatively simple to do, has some issues:
- Low throughput: it is difficult to analyse multiple proteins simultaneously
- Limited cross comparability: since antibodies to different proteins can have different affinities, they cannot be compared with each other.
- Low sensitivity
- Not very quantitative
LCMS addresses all the above limitations of western blot. It is also possible to do targeted proteomics i.e. study just some few proteins instead of the entire proteome. An LCMS system has a high initial setting up cost but a relatively lower run cost. However, the mass spectrometer has to be maintained and it is not an easy job; you basically need a facility and a dedicated technical staff.
Which one would you recommend and would you do both for validation?
Which technique to use depends on what you really want to see and what resources you have at hand. It is a commonly practised approach to do a western blot for re-validating a few selected genes identified from LCMS experiment, which is just to prove that the result can be obtained using another technique too.
Although quantitative methods using MS have been developed, MS is not inherently quantitative. Quantification with MS could be quite tricky. Therefore, it is not the first choice. But, if you do not know which protein levels change and want to find proteins the expression levels of which are different between your samples you are going to compare, MS is not bad idea. In this case, validation is necessary. In other words, MS is not used to validate WB results in general.
In your case, you know which proteins you want to see. Therefore, I would suggest WB. This is a more direct way. Validation of WB may not be necessary, but it is better to get some supportive data: the protein activities in lysates, mRNA levels, etc.
The best way is to use FPLC if you know what kind of protein you're looking for.in case you don't know what are you looking for,then you can run a 2D-PAGE and after analyzing spots then use LC MS/MS to identify your proteins and then continue with FPLC ( for record FPLC is a method of HPLC or LC which is protein friendly and even you can use it to isolate and purify your chosen protein )