Frequently Asked Questions – FAQs

How much antigen do I need?

For one epitope mapping, minimum 10 nmol of protein. If your antigen is 50 kDa, you need 500 μg of the protein. For subsequent epitope mapping of another antibody against the same antigen, less protein may be needed.

What should the antigen sample concentration be?

Minimum 10 μM, preferably 20 μM or higher. If your antigen is 50 kDa, the concentration should be at least 0.5 mg/mL.

How much antibody do I need?

We typically use 2 mg of antibody for immobilization.

What should the antibody sample concentration be?

Minimum 1 mg/mL, preferably 2 mg/mL or higher.

Fab or IgG?

Either works fine.

How pure does my protein have to be?

We usually ask the purities of the antigen and antibody to be better than 80% by LCMS. If you do not have LCMS analyses of the protein samples, ExSAR will perform the analysis with extra cost. In general, the purer your samples are, the higher your chances of obtaining good results.

What do we have to provide?

Your antigen, the exact sequence of your antigen construct, the locations and types of PTM if any, the locations and linkages of disulfide bonds if any, and the conditions which your antigen is in (buffer, salt, additives etc). Your antibody and the conditions which your antibody is in (buffer, salt, additives etc.). We do not need the sequence information of your antibody.

What kind of buffer conditions can I use for my antigen sample?

Most common buffers, salts and additives are OK. Some exceptions are detergents with oligomeric ethylene glycol chains, such as Brij or Triton. For a complete analysis, the buffer components must be listed when the sample is sent.

What kind of buffer conditions can I use for my antibody sample?

Most common buffers, salts and additives are OK. Exceptions are amine based buffers and/or additives, such as Tris.

What kind of exchange conditions can I use?

The exchange reaction is initiated by adding the exchange buffer to your protein sample. The exchange buffer can be the same, or different than the protein buffer. For the exchange buffer, most common reagents, salts and additives are OK. Some exceptions are detergents with oligomeric ethylene glycol chains, such as Brij or Triton. If you want the exchange reaction conducted in conditions other than our standard (which is PBS pH 7.0), please provide the exchange buffer. We can help you decide on the best exchange conditions. We also offer two standard exchange temperatures at 0 °C and 23 °C. Wide applicability is one of the strengths of ExSAR’s H/D-Ex technology.

How much does the analysis cost?

It depends on the size of your protein and PTMs. Please email busdev@exsar.com to request a quote.

How shall I ship the sample?

The sample can be in solution, frozen solution or lyophilized. If you send a lyophilized sample, please include the reconstitution buffer.

My antigen has disulfide bonds. Is it going to be a problem?

Usually no, but the location of the linkages should be provided.

My antigen is phosphorylated. Is it OK?

It is OK, as long as you know where they are and let us know. If you do not know the sites, we can search for them, at an extra cost.

My antigen has glycosylation sites. Is it OK?

It is OK as long as you know where they are and the add-on weight at each site. If you do not know the molecular weights of glycosylation, we can still do H/D-Ex of your protein, but we may not be able to obtain the information around the glycosylation sites.

Are there any good references?

For technology: Y Hamuro, et al. Rapid Analysis of Protein Structure and Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry. J Biomol Tech. 2003;14:171-182 and references therein.

For construct optimization: D Pantazatos, et al. Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS. PNAS. 2004;101: 751-756.

For protein-ligand interactions: Y Hamuro, et al. Hydrogen/Deuterium-Exchange (H/D-Ex) of PPARγ LBD in the Presence of Various Modulators. Protein Sci. 2006:15;1-10. Y Hamuro, et al. Application of Hydrogen/Deuterium-Exchange to p38 Mitogen-Activated Protein Kinase. Amer Biotechnol Lab. 2007;February:28.

For protein-protein interaction: J Horn et al. The role of protein dynamics in increasing the binding affinity of an engineered protein-protein interaction established by H/D exchange mass spectrometry. Biochemistry. 2006;45:8488-8498.

For epitope mapping: SJ Coales, et al. Epitope Mapping by Amide Hydrogen/Deuterium Exchange Coupled with Proteolysis and Liquid Chromatography Mass Spectrometry. Rapid Communications in Mass Spectrometry. 2009;23:639-647.