I ran an SDS-PAGE gel of my total cell samples, each expressed in increasing concentrations of a co-factor to see whether this improves expression of the target protein. I coomassie stained the gel and it showed 2 of the lanes are more intense than the rest, as in the entire lane and all the bands are more intense. Is this due to increased expression of protein or is it a loading error, the lanes were loaded with equal volumes and the optical density of the samples was measure prior to loading- they all had around the same OD ~1.25.
I am quite new to labs so am unsure how to interpret SDS-PAGE gels.
Upon western blotting the gel, there was increased intensity of band of the desired protein in the lanes with more intense coomassie stain. Since it wasn't just the protein of interest band that was more intense with coomassie, i'm guessing the protein of interest expression wasn't increased.
Hi, i'm gonna try to help you as much that i can :).
First: Be sure to load the same quantity of total proteins on your SDS-PAGE.
What do you mean by "the optical density of the samples was measure prior to loading- they all had around the same OD ~1.25." ?
This is the absorbance of your sample at 280 nm ? Have you done some Bradford assays to know the exact concentration of each samples?
Next: If you have more total proteins detected (in your Coomassie) for the lanes of interest you can't compare the signals of your Western-blot.
In fact, for me you have probably loaded more proteins in your lanes of interest.
I hope my answer can help you !
The first thing is to take the same amount of cells then use the same amount of buffer for lysis then if you are adding a cofactor in some samples ( Like if you add 2 microliter cofactor in sample A then in sample B you should add buffer) then only you treated all samples equally. If you did this then you are fine. Then you can also do Bradford and load an equal amount of protein ( 50 to 100ug total load is fine )
Then suppose you have more expression in 2 lanes then indeed you have more expression in those two conditions or you have a loading error. You can see multiple bands may be because your protein is degraded. Do you also find many bands in western blot?
Ideally, you should see the thick band of your protein of interest. It is possible that a few other bands will appear.
The very crucial thing in such kind of experiment is to load control means do everything same but don't induce
If Coomassie stain shows higher total protein content, it is obvious, that you loaded more proteins in these lanes. For the Western blot application, you should load the same amount of protein in each well, so from your results, it would be rather difficult to derive any convincing conclusion about the treatment effect.
I would suggest checking your sample buffer: many components used in lysis buffers might absorb light at 280 nm (for example RIPA buffer and TritonX-100 do absorb significantly). If you use such buffer and do not blank the spectrometer (photometer) against the buffer, then you can't say what is protein concentration in your samples (for example if you have ~1.25 OD, it may be buffer who gives 1.23 units, while proteins give the rest; then 1.24 and 1.25 OD would mean 2 times difference in protein concentrations).
So, just measure OD 280 for pure buffer. From this value, you might estimate the actual ratio of protein concentrations among the samples.
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