Q: How much protein and what purity is needed for protein sequencing?
A: We typically accept samples at 100 µg with at least 80% purity, rare in the field.
To assess purity of your samples, you may first start by running your sample on a reducing SDS-PAGE acrylamide gel. If you would like to send an antibody, you should be able to see two distinct bands at 25 kDa and 50 kDa for the light and heavy chains, respectively, after staining the gel with Coomassie. However, if you have additional antibodies other than your target antibody in your sample, these contaminant antibodies may interfere with the protein sequencing of your target protein. See our blog on common contaminants in protein sequencing for more information.
If you are unsure about the quality of the gel, please send us a picture and we will provide advice on how to proceed. If you are unsure whether one or more antibodies are present as contaminants in the sample containing the antibody you wish to sequence, please contact us as well and we can discuss. If you would like to learn more about the methods used to assess the purity of proteins, check out this resource: Raynal et al. (2014) Microb Cell Fact 13: 180
To purify your protein, you may use affinity chromatography with binding proteins such as antibodies against your target protein, or antibody-binding proteins like protein G, protein A, protein L or other available commercial slurries. Here are two resources on protein and antibody purification:
- Protein purification: Ritchie (2012). Mater Methods 2: 134
- Antibody purification: Rodrigo, Gruvegard and van Alstine (2015). Antibodies 4(3): 259-277
At Rapid Novor, we specialize in mass spectrometry (MS)-based sequencing of proteins, and particularly antibodies. To perform protein sequencing, we utilize liquid chromatography tandem MS (LC-MS/MS) instruments that are high-resolution and high-throughput. Our research core has more than 20 years of experience in mass spectrometry, protein chemistry and machine learning. We are sure we can handle any challenges you may throw our way, so please contact us to see how we can fit your needs.
|Maria Rosales Gerpe, Ph.D|
Rapid Novor, Inc.