Good afternoon,
We're having trouble running the Bradford assay for analyzing proteins. We are hitting a wall trying to produce a curve, and we decided not to analyze any samples before solving that problem.
We are using Sigma's Bradford reagent (B6916; https://www.sigmaaldrich.com/catalog/product/sigma/b6916), and made several attempts to construct a curve using protein standards (BSA). In all cases, BSA was diluted in PBS, incubated for 20-30 minutes under protection from dark (using aluminum foil to protect the microtubes from the light), and vortexed before reading. Samples were read at 595 nm first, and then at 450 nm; the idea was to linearize the curve by analyzing the ratio between absorbances at these wavelengths. The protocol varied as we attempted to understand what was going on, and these are the results:
First attempt
1.5 ml Bradford
200 ul sample (BSA+PBS)
Incubated for 30 min
Four readings from each point
Concentration:Average absorbance at 595 nm:Average absorbance at 450 nm
0 : 0 : 0
19.95 : 1.62625 : 0.06175
40 : 1.88875 : 0.010125
60 : 0.90625 : 0.04025
80 : 2.13475 : 0.0535
100 : 2.06225 : 0.094
120 : 0.47 : 0.04825
Second attempt
1.5 ml Bradford
200 ul sample (BSA+PBS)
Incubated for 20 min
Four readings from each point
Concentration:Average absorbance at 595 nm:Average absorbance at 450 nm
0 : 0 : 0
19.95 : 0.78925 : -0.20694
40 : 0.62323 : -0.20094
60 : 0.568 : -0.20697
80 : 0.87698 : -0.20703
100 : Not made
120 : Not made
Third attempt
1.5 ml Bradford
200 ul sample (BSA+PBS)
Incubated for 20 min
Four readings from each point
Concentration:Average absorbance at 595 nm:Average absorbance at 450 nm
0 : 0.301 : -0.275
19.95 : 2.058 : 0
40 : 0.792 : -0.04
60 : 2.031 : 0.001
80 : 2.22 : 0
100 : 2.204: -0.001
120 : 2.228 : 0
Fourth attempt
900 ul Bradford
500 ul sample (BSA+PBS)
Incubated for 20 min
Four readings from each point
Concentration:Average absorbance at 595 nm
0 : 0.0935
19.95 : 0.15467
40 : 0.20233
60 : 0.20933
80 : 0.23633
100 : 0.219
Fifth attempt
1.9355 ml Bradford
64.5 ul sample (BSA+PBS)
Incubated for 20 min
Four readings from each point
Concentration:Average absorbance at 595 nm:Average absorbance at 450 nm
0 : -0.002 : 0.101
19.95 : 0.002 : 0.5275
40 : 0 : 1.0335
60 : 0.0025 : 1.032
80 : 0 : 1.0295
100 : 1.0235
Sixth attempt
900 ul Bradford
100 ul sample (BSA+PBS)
Incubated for 20 min
Four readings from each point
Concentration:Average absorbance at 595 nm
0 : 1.295
19.95 : 1.153
40 : 1.195
60 : 1.148
80 : 1.181
100 : 1.148
As you can see, there is a systematic error in which, in all attempts, there is always a point in which absorbance is lower than that of the previous point; these are marked in italics above. We don't know what we are doing wrong! Help!
The Bradford protein assay is the easiest assay known to man. I can't tell for sure what your problem is but follow the instructions for Catalog #B6916. Don't deviate. The ratio of reagent to sample is supposed to be 30:1 but it varies all over the place in your attempts. And you should have great linearity just using the 595-nm wavelength reading. What are the the units for your standards? Standards should be ~1.4 mg/ml or less. Hopefully, you are not using 20-100 mg/ml.
We've experimented with the instructions from Sigma and, not having a result, attempted to use different dye ammounts. The concentration range are in the 0.2-1.2 mg/ml range.
Dear Caio,
depending on the spectrophotometer used, absorbance readings above 1.2 may not be reliable or reproducible. Two things also need to be considered:
1. of all the protein spectrophotometric analysis, Bradford is one of the most sensitive (more than Lowry's method for example)
2. all standard curves, although initially linear, from a certain high concentration they bend, starting to become horizontal.
Perhaps this may be explaining what is happening. My recommendation would be to use lower concentrations to perform the standard curve with absorbance results up to 1, or to use another method such as Lowry, which is slightly less sensitive and better suited for higher protein concentrations.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running