FAQs for Proteomics Services
Q1) What is the best method to identify my protein?
Ans: We strongly recommend mass spectrometry (MS/MS) for protein identification and for internal sequence information. With an appropriate strategy, you may sequence the entire protein. This technique can readily provide sufficient data to show homology with other proteins, or for novel proteins to obtain internal amino acid sequence information to enable cloning of the gene.
The alternative approach of traditional N-terminal Edman however has the benefit of providing the definite sequence of the N-terminus. For the proteins that are blocked at the N-terminus, MS/MS will be the best option to generate sequence data.
Below is the schematic flow to assist you to choose the method that suit for identifying your proteins.
Q2) How do I submit my samples for protein identification?
Ans: Most of the proteomic services can accept pure protein in dried format.
For MS/MS service, it is more convenient for the customers to send the gel slides in 1.5mL of PCR tubes directly. There is no buffer/ water required to merge the gel slides. Pooling and duplicates of samples are necessary according to the type of staining.
Below is the quick guide of the sample submission for protein identification services: -
||Gel Slice from SDS PAGE
|Recommended for Pure protein: 100ng to 1ug, in < 250uL.Solution needs to be freeze-dried or air-dried in 1.5mL tubes.
Free of interfering buffers and salts.
Buffers containing primary amines, such as TRIS and HEPES, should be removed. Low concentrations of SDS (up to 0.3%) and PBS can be tolerated
|Yes. Excised into 1.5mL tubes and dried.
NOTE: no water or buffers required.
Pooling of 2 spots (each spot contains minimum 100ng): Stained with Coomassive Blue; or MS compatible Silver Stain (within 1 month of preparation).
Pooling of 4 spots: Stained with SYPRO Ruby or Cy dyes.
|De Novo Protein Sequencing
|N-terminal Protein Sequencing
||Yes. Send blot on sequencing grade PVDF membrane (do not excise), Enclose a photocopy of membrane or gel with band of interest marked out.