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.
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.
@VanceAlbaugh The only advantage of western blot is that it is easier to do and most people are comfortable with it. As you said, it is certainly cost-efficient and easy when only a few proteins are to be studied. However, even in this case cross comparisons are difficult. Antibody-protein interactions for different pairs are not comparable. It can be much more biased than fragmentation/ionization bias in MS.More
Peptides are ionized to determine the mass and ionization efficiency is not predictable. It would depend on peptide sequences and probably environments surrounding peptides. I think the word spectrometry in MS is used because you can see spectrum of molecular mass.More
(test vs treated). At least I know this is so for SILAC and ITRAQ. I said standard curve is necessary for WB because there is no way you can compare two different antibody labels from the same blot. This is turning into a huge discussion and perhaps we should discuss this in chat.More
; the name itself implies quantification. When you say it is not so quantitative, what are you comparing it with?More
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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.
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.
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.
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Western blot, though is a commonly used technique and is relatively simple to do, has some issues:
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 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.
Western blot, though is a commonly used technique and is relatively simple to do, has some issues:
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 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.
More
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