After adjusting concentration of whole cell proteins from different bacterial strains, I want to measure relative expression of a particular protein by Western blot (DAB, Diaminobenzidine method). First I stain SDS-PAGE gels with coomassie and then take image with Gel Logic 1500 Imaging System (Kodak), choosing 16 or 8 bit TIFF image. I am using Image J to measure band densities, for few selected bands, to measure and normalize amount of whole cell proteins to be loaded into the wells. As such there is no known protein to serve as an internal control for this bacteria, therefore I randomly select few protein bands. Is it an acceptable method by peer reviewed journals?
Dear Muhammad,
Although I have no formal research training in microbiology, but from the experiences of my graduate studies with human cell lines, all I would like to suggest you that, why don't you go for a spectro-photometric measurement of your protein samples, prior running them in the SDS-PA gel. All you need to do is to just use some plastic cuvettes and a calibrating reagent (which is widely available from Bio-rad and Fermentas).
By this method you can effectively and scientifically quantitate all your protein samples, by taking BSA as the standard.
This process is not only widely acceptable among scientific communities but also a convenient one too.
Once you have successfully quantified all your samples, then you can load them into SDS-PA gels as per protein concentration (say 50 ug for a small gel or so).
Then I think you will not be facing the problem of in gel quantifications and other related complications. Then you can take any protein of your choice and knowledge, that is known have no effect on its expression, and consider it as standard, to show a steady pattern of bands in all the lanes, which will demonstrate an equal protein loading of all of your samples.
Dear Chatterjee,
Thanks for your response. The strains I am studying are wild-type, its mutant, and some transformants of these two strains with different plasmids. So the transformants would be expressing some proteins in abundance as compared to untransformed wild-type and its mutant. In this case, I assume, whole cell protein concentration determination may not be suitable to normalize their loading volume. In such cases mostly people use internal control, which doesn't change its concentration under different conditions, but I don't have such internal control for my bacteria.
One disadvantage of relying only on spectrophotometric quantification of total protein abundance in a lysate is that it in facts only tells you the amino acid content of your sample. However, some of this may be degraded. People typically first quantify protein sample (Brandord, BCA or similar methods), load equal amount of protein per lane based on this, but then also verify equal loading either by staining the gel (or membrane after the transfer) and/or by looking at expression of house keeping genes. This is done best by western blot. You can also use the same number of cells per sample. Muhammad, are you trying to compare expression of your overexpressed proteins? Or the effect of overexpression on an endogenous protein?
Dear martin Holcik, Thanks for your detailed answer. I'll now definitely use some quantification kit first. I am basically comparing a mutant (temperature-sensitive strain) Mycoplasma vaccine with its wild-type (non-temperature sensitive). My previous whole genomic comparative study has revealed a mutation in a GTP binding protein (Obg).
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0073954.
Now I am trying to further characterize effect of this mutation on growth or any other phenotype. I also complemented this mutant with wild-type copy of this gene. During expression analysis of this protein among wildtype, mutant and their transformants with and without obg containing plasmids, a differential expression of Obg was found. Difference of expressin between wild-type and its mutant vaccine is more of interest. So, I am just trying to validate this differential expression by some suitable and scientifically valid method. There is known housekeeping gene that have been used for mycoplasma for such analysis.
Muhammad, in that case you can also compare mRNA levels of Obg in each strain by RT-qPCR (again relative to several housekeeping genes).
It depends on the coclusion you would like to give. You can adjust all samples to a constant total protein content if you would like to show, that your proteins was changed. You can adjust your sample to a constant house keeping protein and you can do this also vv. adjusting to a constant concentration of your protein ... to demonstarate that anything else around was changed.
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