Hi there
I have been doing microplate bradford assays on protein samples (they are relatively crude). I have used BSA as a std and each time the standard curve is consistent in slope and intercept from day to day.
However, something odd is happening with my samples.
I had no idea what concentration to expect so I did a bradford on the original, 2-fold and 5 fold diluted sample and they fell within the std curve.
When i do the calculations and multiply by my dilution factors I get VERY different concentrations. i.e. original: 0.8mg/ml, 2 fold: 1.3mg/ml, 5 fold: 2.1mg/ml.
I am confident in my pipetting and dilutions (I also repeated everything with the same result). I looked into interfering agents, but my samples have no detergents in them.
The proteins are in 10mM MOPS PH 7.0, 100mM NaCl, 1mM DTT.
Any help would be much appreciated!
you can go for lowry method for protein estimation which is simple and reliable method
First of all Bradford's method produces a non-linear response. Second of all, it is an approximate method. Do not expect to much from it.
It would be helpful if you would share your standard curve and the raw data for the 3 samples.
Generally, all the assay methods are relative. How about instead taking original, 2-fold and 5-fold dilution you stick to just one. Take suppose 1microlitre of the sample and replicate it three times. If your value is getting replicated then stick to this only. In this way you will not get the confusing results that you are getting due to various dilution.
Hope this helps.
Hi,
are You sure that You are in the right dimension? For me Bradford is stable until 20 µg/ml and You say You have 800 µg/ml.
Steffi
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