I have measured protein concentration using nanodrop 1000 at 280nm where I took 1ul of samples and took lysis buffer as blank.
The software shows 1 abs =1 mg/ml .
And I have got nanodrop values like -0.83 mg/ml .
I want to know what does the negative sign indicates ?is there proteins or no proteins that's why it is negative.I could not do Bradford assay because of some technical issue with plate reader.kindly help what does the negative sign indicates.
The machine compares the absorbtion of your blank, with your sample. Because the only thing different in your sample is the protein, the resulting difference in absorbtion is reflecting your protein. If your measurement is negative, it would suggest, that your blank has a higher absorbance, than your sample. So maybe you have mixed these two up, forgot to blank or used the wrong blank. It could also be that you had bubbles in your sample.
The recorded A280 may be in a negative range. Use an appropriate blank (Buffer) to get a positive value for the test sample containing protein.
Well, your sample has a lower absorbance than your blank, so there are several explanations :
You haven't used the proper blank, your blank is contaminated, or your protein concentration in your sample is too low to make a difference. Also perform the measures in duplicate or tripliate if possible, and keep in mind that you can get bad absorbance values from bad pippeting (bubbles during measure, incorrect volume etc.)
Try two different blanks. In my experience, the Nanodrop is not too reliable for measuring proteins at low concentrations.
In my experience, this happens when the blank you're using was made differently from the buffer you have your protein in. I usually use the very same buffer I dialyzed in as my blank. Another option is to use a clean concentrator and use the filtrate as your blank, but this works only if your protein is very pure.
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