protein concentration - (Dec/03/2008 )
I have encountered into a problem concerning protein measurement.
I have two different protein samples: from roots and from leaves. I have extracted proteins from similar amount of sample but got more out from leaves than roots measured by Bradford. Based on Bradford measurements I have precipitated same amount of protein with cold acetone. After centrifugation the pellet in root samples is much bigger than in leaf samples althought I think they should be same size if same amount of protein is precipitated. I wonder if Bradford is not able to compare protein amounts from different sample origin or is something else is also precipitated in root samples. I havent been able to measure protein samples after precipitation and resuspension as I have resuspended the samples into sample buffer. I should load equal amout of protein to the gel for Western blotting and dont know what to do now. Has anyone encountered into similar problem and would have some advise to give.
It's a problem for every measurement of protein, every protein is a protein but they are all different and will give therefore each a different result. only if the sample and the standards contain the same protein(s) you can be sure about the outcome. In the other cases there is uncertainty about the results. But in your case I think it's more probable there are different proteins in root and leaves and for the loading of the western I would use the results of Bradford because it's the best your got.
I now measured protein concentration A280. The results are strange. As I will get double the amount of protein in leaves compared to roots with Bradford method, A280 measurement says that there is double the amount of protein in roots compared to leaves. So the results are totally opposite. The results are similar even after precipitation of the proteins with acetone (I left bromophenol blue off). Maybe I should try another kit to measure protein concentration. Would Lowrys method be good?
It's as good as Bradford or A280 you get results equal to Bradford, A280 or different but there is no way to tell wich method gives the best result.
I would use de Bradfort results to go on for the same reason that I can't tell wich is the best result.
Maybe if I tought it's crucial and the sample is 90% or more protein I would dry freeze the protein and go by weight
Thanks for help.
I also think that Bradford is more reliable than A280. It is posible that something else in addition of protein are precipitating in root sample. Do you have an idea what can cause this and how to get rid off it? Maybe changing buffer pH? The pellet is hard to dissolve when it so big and if I predipitate lower amount, the protein concentration is not enough for the gel.