I have a protein which shows a molecular mass of approximately 15 kDa under reducing conditions in SDS-PAGE. Under non-reducing condition, it shows three bands with molecular mass of 27 kDa, 52 kDa and 105 kDa, suggesting it is probably a multimer (?). MALDI analysis of the protein shows peak at 26.9 kDa and 53.5 kDa (no peak is observed for 15 kDa and 105 kDa subunits). What molecular mass should be considered for calculating its molarity?
If your monomer mass is 15 kDa but you are uncertain about the oligomer mass, or the oligomer is heterogeneous, I recommend using the monomer mass to calculate the concentration, and base all subsequent experiments on the concentration of monomer.
It sounds to me that you have a monomeric MWT of 15 kDa and your protein of interest likes to be in a dimeric and tetrameric state (under your testing conditions). I assume that the SDS-PAGE analysis shows one clearly defined band with little smearing... I am guessing that you are a little confused due to the MALDI results. Perhaps you can try running MALDI under slightly different conditions to convince yourself that the 15kDa species is your monomer. Furthermore, if there are known homologues of this protein, you can certainly perform a literature search and identify the likelihood of higher oligomeric states. http://www.ncbi.nlm.nih.gov/cdd/
Dynamic Light Scattering and Small Angle X-ray scattering (SAXS) will allow for a more thorough experimentation of the oligomeric state of your protein in different solutions. Have you run this sample through gel filtration? It may be advantageous to establish if concentration plays a major role in the oligomeric state of your protein of interest. I suspect that you would find evidence for a monomer, dimer, tetramer, and octamer with each abundance potentially affected by buffer concentration, protein concentration, pH, and salt concentration. In the past I happened upon a protein that demonstrated a concentration dependent dimerization at concentrations that exceeded physiological conditions. If I took the monomer fraction from the GF experiment, incubated on ice for a few hours, and re-injected the sample I got the same oligomeric distribution from the previous run.
Best of luck.
If your monomer mass is 15 kDa but you are uncertain about the oligomer mass, or the oligomer is heterogeneous, I recommend using the monomer mass to calculate the concentration, and base all subsequent experiments on the concentration of monomer.
So, what your saying is:
Your protein has a monomeric weight of 15kDa, and it forms disulphide bonds with itself to give a dimer (27kDa), a tetramer (52KDa) and a heptamer (105kDa). This can sometimes be an artefact of SDS-Gels. Recall APS is classified as an oxidant!! The MADLI confirms the existence of a dimer and tetramer, but this was presumably under non reducing conditions ( is it also denatured?).
You need a native technique such as gel filtration, analytical ultracentrifugation or even native mass spectrometry to confirm if the protein is a monomer, dimer, etc in solution.
Do you not have some kind of clue from the protein sequence?
I have a question: Have you reduced your protein previously to MALDI determination? Like in the previous treatment to SDS-PAGE where you can observe a 15 kDa band.
MALDI MS is much more confident to MW determination than SDS-PAGE. I remember in my thesis job the difference between SDS-PAGE and MALDI MS MW determination was of some few kDa for a nominal MW of 24 kDa. After to clone it I could confirm that MALDI MS was right. So usually I trust over MALDI in instead of SDS-PAGE to determine MW of proteins.
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