I have isolated a plant protein fraction forming four MW closely-related bands in SDS-PAGE, ranging 30-35 KDa. In the absence of 2-ME, the fraction resolved as 2 bands with MWs of about 28 and 30 KDa. Native PAGE using 8, 10 and 12% PAG (pH 8.8, Laemmli system) showed 2 protein bands with MWs of about 65 and 68 KDa. So, what explanation do I have for protein subunit conformation? What is the simplest way for separation of these proteins?
Thanks
The simplest case is a single protein with various degrees of post-translational modification and proteolysis, which is a dimer under native conditions and contains one or more intramolecular disulfide bonds. Have you attempted to identify the bands in SDS-PAGE by mass spectrometry of tryptic peptides and comparison to genomic information?
It depends how you isolated that protein fraction. If the last step of your purification procedure was immunoaffinity chromatography, then all your four or two proteins might be immunologically similar to each other. If this is the case, the most likely difference between them is some post translational modification like glycosylation. Since in the absence of 2-ME the fraction resolved as 2 bands with MWs 28K and 30K, they are not some kind of disulphide dimers. The native PAGE data showing the MWs ~ 65K and 68K does not indicate that the proteins are non-disulphide dimers. Usually, the natural non-disulphide dimers are pretty stable in 1% SDS-Sample Buffer without boiling in regular SDS-PAGE. If your last step of purification of this protein fraction was not immunoaffinity chromatography, you might want to consider to run western blot under reduced and non-reduced conditions using some antibody (if available) to any protein in this mixture. With positive results you might use this Ab for preparation of affinity column and purify your protein by immunoaffinity chromatography. If no Ab available, you can try to use hydrophobic or ion-exchange chromatography to separate these proteins. There is a general, mostly empirical rule in protein purification: any two different proteins from the same species can not have identical (or very close) molecular weights, hydrophobic properties (behavior in hydrophobic chromatography), and the charge (behavior in ion-exchange chromatography). There was an old computer game on teaching strategy in protein purification from Pharmacia company that demonstrated that rule in practice.
Hi,
Yeah its interesting.. Do you have clear perfect protein band/s? Single protein band is must in the isolated sample while running in native PAGE. Then how do you know about its molecular weights in native PAGE, how you will accept native molecular weight estimation by molecular weight markers. Because PI value of native protein as well as net charge mass of native protein will differ from their subunit/s of SDS-PAGE so then how do compare native PAGE profile with SDS-PAGE. Do molecular weight estimation by suitable column of Pharmacia or other source with HPLC system then get perfect molecular weight estimation after purifying the native molecule by any one method. Go through the Pharmacia Protein Purification manual/s and find suitable method to isolate single protein band in native PAGE as well as in HPLC, through purification test experiments.
I know protein purification from the plant system is very difficult even though we can get success by hard work and trials.
I wish you all the best.
sakthi.
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