Sample buffer compatibility
Besides proteins there are a huge number of other components in the samples intended for total protein concentration measurement. There are a few types of total protein assays on the market, and they all show different sensitivity to proteins and different incompatibility to sample components.
The ultrasensitive Dot-it-Spot-it Total Protein Assay shows less interference to components, and higher sensitivity to proteins, compared to presently available assays.
We have tested the measuring range for BSA with the buffers described below. The buffers have also been tested with seven other proteins. The list will be updated.
A few sample buffers can be applied directly to the Detection Sheet, but for most of the compatible buffers it is recommended to dilute 50% (1+1) with our Dilution Buffer.
1. Sample buffers that can be prepared with up to 50% sample
Measuring range for BSA down to 0.2 μg/mL using 50% sample in the application mixture.
TBS: 20 mM TRIS buffer pH 7.5, 0.15 M NaCl, 0.02% NaN3.
Tested additives to TBS:
– 0.03% Tween 20
– 10 mM EDTA
– 2 M Urea
PBS: 20 mM phosphate buffer pH 7.5, 0.15 M NaCl, 0.02% NaN3.
If you dilute to 10% sample buffer in the application mixture, you can use 5 times more of additives and measure down to 1 μg/mL.
2. Special buffers allowing 100 % sample
Measuring range for BSA: 0.15-15 μg/mL using 100% sample in the application mixture.
Laemmli sample buffers (1x) mixed with PBS (0.5x):
– Biorad Laemmli:
33 mM TRIS, 13.2% glycerol, 1.1% SDS, 0.005% BFB, 5 mM phosphate, 75mM NaCl.
– Sigma Laemmli:
62,5 mM TRIS, 10% glycerol, 2% SDS, 0.004% BFB, 5 mM phosphate, 75 mM NaCl.
3. Limiting sample buffers
Some sample buffers interfere with the kit reactants but works well when a lower amount of sample is used in the application mixture.
RIPA buffer Pierce: 25 mM TRIS, 150mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS.
Max 4% sample mixed with Dilution Buffer in the application mixture
Measuring range for BSA: 2.5-250 μg/mL with 4 % sample.
Researchers must validate the compatibility to decide if the assay is acceptable for their specific purpose.
BFB: Bromophenol blue
BSA: Bovine serum albumin
DOC: Deoxycholic acid
SDS: Sodium dodecyl sulphate