The recombinant protein 18kDA has been purified by His Tag and also run on a gel filtration column where it appears in the void and comparing with standards appears between 158000 Kda and 67000 Kda.Can I proceed with NATIVE PAGE to confirm the exact size with a marker.
Hi Gautam,
from my experience, proteins run on a Native PAGE not just according to their size, but also to their shape and this is a very important parameter tha you have to consider. In fact, if your protein ran between 158 and 67 KDa on a size exclusion, there are chances that you get the same result on a Native PAGE. there are several ways to investigate the size ( SAXS, RALS, DLS, SLS,..) but unfortunately the calculation of the MW is made considering a globular shaped protein. The most accurate way that I would suggest you is an analytical ultra centrifugtion.
Goodluck
Native PAGE does not allow to determine the protein molecular weght.
which gel have you used for your studies: sephadex G200, Sephacryl s300, ...?
you must use the proper gel for your protein.
If you like to do the experiment with the small amount of protein, please try to explore our method using thin layer gel filtration method on Sephadex or other printed in 1971 and in 1972: Z Wasyl, E. Luchter, Bielanski W Jr, both in BBA. I am on vacation and I do not have citations nor pdf with me.
I have used that method for my work on subunit structure of PAP, 1974, BBA, E. Luchter-Wasyl and W Ostrowski.
please look that papers. maybe they will help you.
If you must use native page, see the article linked below and the several articles it references in the introduction. These methods require several gels of different densities to distinguish contributions from charge from those associated with size/shape. (See also Blue Native PAGE)
See: http://doi.org/10.1002/elps.1150091203
If you have access, you may prefer Multi-Angle Light Scattering (MALS). Unlike DLS it does not require assumptions about shape and it is somewhat more accessible to a generic biology/biochemistry lab than Analytical Ultracentrifugation (AUC).
http://doi.org/10.1002/elps.1150091203
In Native-PAGE， one of the problems that cannot be neglected is the pI of a protein, that means the different charge of the protein, If your loading buffer not contains SDS. Thus, except for protein size, the different charge might also contribute to a different migration rate, ???
In contrast to SDS-PAGE (in which proteins are completely denatured with SDS and a high concentration of negative charges is attached to them) gel electrophoresis under native conditions depends on the existing charges of the protein. Therefore, the selection of the buffering system depends on the isoelectric point of the protein. A protein with a pI value of 6.2 is weakly negatively charged in a buffer with pH 7 and therefore migrates more slowly to the anode than another protein of pI 5 in the electric field. Strongly basic proteins (with a pI value above 8) can migrate in neutral buffers by exchanging the anode and the cathode.
In addition to the charge of the proteins, their size and shape also influence the migrating properties. In order to minimize these factors, polyacrylamide gels of low concentration and cross-linking are often used, which on the other hand reduces their mechanical stability and makes handling more difficult.
In order to keep the proteins to be separated folded, the buffer must be adapted to the requirements of the proteins (reducing agents, etc.) and, if necessary, cofactors (nucleotides, cations, etc.) required for stability and activity into the buffer or even into the buffer Gel.
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