In my thesis I will need to compare rubisco and rubisco activase content at protein level. Can I use SDS-PAGE for comparison of rubisco and rubisco activase content in two samples or I must use western blot?
Hi there,
I guess you intention is to use gel staining for protein quantification. The problem is that the staining efficiency does vary from protein to protein. So the accurate quantification using staining involves the calibration of the staining for each protein (ie having the pure protein available in order to measure the relation between the intensity of the band staining on gel and the quantity of protein loaded).
The problem will be the same if you use WB and specific antibody for each protein : you will need to calibrate the signal obtained towards the quantity of protein loaded.
The only simplier case is the one with both proteins are tagged with the same tag and therefore relative amounts of proteins could be deduced directly from WB band intensities (using anti tag antibodies).
If the molecular weights of all the polypeptides are sufficiently different that they can be separated on SDS-PAGE, you could, in principle, quantify each of the proteins, assuming you had a standard, as Dominique pointed out. A difficulty that could arise is if the amounts of the two proteins are vastly different, so that you would have to overload the gel with one of them to be able to detect the other. The band for the large subunit of Rubisco would probably overwhelm the band for activase, making quantitation impossible. Therefore, this method is only useful if the proteins are present in similar amounts, let's say within an order of magnitude on a weight basis.
I don't need absolute content of rubisco. I want to resolve them by SDS-PAGE and then measure (guess) rubisco relative content by BSA standard curve after gel extraction of rubisco. For RCA, I am trying to find a good extraction protocol.
You can run SDS-PAGE and then use densitometer or some intensity evaluator software for rough comparison.
I know people do that for Rubisco. As it is the most abundant protein in a leaf extract, you don't need antibodies to identify it, and SDS PAGE is good for semiquantitative comparison. Not sure if you can identify activase in the same way though.
Yes you are correct but many articles used antibody and no one used only SDS PAGE for comparison. Detection of activase is not as simple as rubisco and i must use antibody.
It has been done (and published) in here:
Photosynthetic and growth responses of a perennial halophytic grass Panicum turgidum to increasing NaCl concentrations.
Cheers, Johannes.
how to calculte rubisco content, i have od value but dont know how to calculate
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