Hi Asma,

Validating proteomics data can be done in a number of ways. Western blotting is an accepted standard if you can get a good antibody and your cells express enough of the target protein to be able to detect it. Flow cytometry is also a great option - again dependant on a good antibody. You may also be able to multiplex some of the key proteins by flow cytometry and retain some cellular morphology information and differentiation markers also.

If your proteomic data supports a changed pathway that results in the production of a metabolite, you might be able to test for changes at the metabolite level also.

RT-PCR and transcript level methods are good for complementing protein level results but I would be cautious in saying they validate proteomic data as changes in protein abundance can be independent of transcriptional changes i.e. protein degradation/ stability/ post-transcriptional regulation etc.

Good luck,

Nathan

Dear Asma

In order to differentiate Osteoblast stages, you could selectively evaluate the stages that are obtained from different cultures/grown under say different conditions.  The validation can be done based on 

a. The proteins that matched/tagged the gel

b. Immunoblotting

c. Mass Spectrometry

Whether or not you have identified biomarkers, you could then check how many of the listed proteins have been mapped to biomarkers. There is no steady method, per se.  But you could setup the threshold by the presence of detectable biomarkers associated with the proteins.

You could use PO tools and biomarker entries from genecards.org

I hope this helps

Prash

Can you give us more information about your experiments and your objectives?  For example, it's not clear if you want to obtain validated proteomic data or if you have proteomic data that you want to validate.  Are you experienced with proteomics and need help with with osteoblast handling; experienced with osteoblasts and need guidance with proteomics; or neither of these?

Thanks!

I agree with John

Your provided data is not enough.

Please discuses your aims with sufficient details.

Hi,

Thanks for the reply.

I have already a proteomics data obtained during osteoblasts differentiation and i need to know the best method to validate them.

Is the flow cytometry, or PCR considered as best method for validation?

the best method for the validation of proteomic data is western blotting.

Also, you can use real-time PCR for each detected gene (related to the protein) in the next step.

I recommended the WB as confirmation tool for proteomic identified protein.

Hi Asma,

Validating proteomics data can be done in a number of ways. Western blotting is an accepted standard if you can get a good antibody and your cells express enough of the target protein to be able to detect it. Flow cytometry is also a great option - again dependant on a good antibody. You may also be able to multiplex some of the key proteins by flow cytometry and retain some cellular morphology information and differentiation markers also.

If your proteomic data supports a changed pathway that results in the production of a metabolite, you might be able to test for changes at the metabolite level also.

RT-PCR and transcript level methods are good for complementing protein level results but I would be cautious in saying they validate proteomic data as changes in protein abundance can be independent of transcriptional changes i.e. protein degradation/ stability/ post-transcriptional regulation etc.

Good luck,

Nathan