Fig 1.
Interference of detergent-containing buffers with the Bradford protein assay.
Different concentrations of BSA were assayed with the Bradford reagent under different conditions: Black: control conditions (BSA dissolved in water). Blue: assay with 1 μl of Laemmli-type SDS buffer (2% SDS and 5% mercaptoethanol)/ml of assay mixture. Purple: assay with 10 μl of Laemmli-type SDS buffer (2% SDS and 5% mercaptoethanol)/ml of assay mixture. Orange: assay with 1 μl of RIPA buffer (1% Igepal, 0.5% deoxycholate and 0.1% SDS)/ml of assay mixture. Green: assay with 10 μl of RIPA buffer (1% Igepal, 0.5% deoxycholate and 0.1% SDS)/ml of assay mixture.
Fig 2.
Effect of alpha-cyclodextrin on the Bradford assay.
Panel A: effect of cyclodextrin on the background induced by SDS in the Bradford assay. Different concentrations of Laemmli-type SDS buffer were added to the Bradford reagent, together with different amounts of alpha-cyclodextrin. Black: control (no added detergent). Blue: 1 μl of SDS buffer per ml of assay mixture. Orange: 2 μl of SDS buffer per ml of assay mixture. Brown: 5 μl of SDS buffer per ml of assay mixture. Purple: 10 μl of SDS buffer per ml of assay mixture. Panel B: effect of cyclodextrin on the Bradford assay in the cuvette format (1 ml of assay). BSA was assayed by the Bradford assay in the presence of different amounts of cyclodextrin and of SDS-containing buffer. Black: control (no SDS, no cyclodextrin). Orange: 5 μl of SDS buffer, 1.25 mg/ml alpha cyclodextrin in Bradford reagent. (CD/SDS molar ratio: 3.65). Green: 10 μl of SDS buffer, 1.25 mg/ml alpha cyclodextrin in Bradford reagent. (CD/SDS molar ratio: 1.82). Blue: 10 μl of SDS buffer, 2.5 mg/ml alpha cyclodextrin in Bradford reagent (CD/SDS molar ratio: 3.65).
Table 1.
Effect of the cyclodextrin/SDS ratio on the assay response.
Fig 3.
Comparison of the effects of alpha and beta cyclodextrin.
Panel A: effect of increasing concentrations of cyclodextrin on the background absorbance of the assay. Different concentrations of alpha-cyclodextrin (black curve) and beta-cyclodextrin (blue curve) were added to the Bradford reagent and the interference measured. Panel B: effects of cyclodextrins in the Bradford assay. Different concentrations of cyclodextrins were used in a protein assay containing 10 μl SDS buffer per ml of assay. Black: control: no SDS buffer, no cyclodextrin. Blue: 2.5 mg alpha-cyclodextrin / ml of Bradford reagent (CD/SDS molar ratio: 3.65). Purple: 5 mg alpha-cyclodextrin / ml of Bradford reagent (CD/SDS molar ratio: 7.3). Green: 2.5 mg beta-cyclodextrin / ml of Bradford reagent (CD/SDS molar ratio: 3.17).
Fig 4.
Responses of different proteins in the Bradford assay.
Different proteins were assayed by the Bradford method using the cuvette format (1 ml of assay) under control conditions (panel A) or after denaturation in 10 μl of SDS buffer and in the presence of 2.5 mg alpha-cyclodextrin/ ml of Bradford reagent (panel B). Black: bovine serum albumin. Purple: hen conalbumin. Blue: soybean trypsin inhibitor. Green: hen lysozyme.
Fig 5.
Effect of cyclodextrins on the protein assay in the presence of RIPA buffer.
BSA was assayed in 10μl of RIPA buffer in the cuvette format (1ml). Blue: assay in the presence of 5mg/ml alpha cyclodextrin. Green: assay in the presence of 2.5 mg/ml beta cyclodextrin.
Table 2.
Effect of the cyclodextrin concentration in an assay containing RIPA buffer.
Fig 6.
Protein assay in the microplate format (250μl).
BSA dilutions were assayed using the microplate format. Black: 10 μl of BSA dilutions in water. Green: 5 μl of BSA dilutions in SDS buffer, reagent made by adding 10 μl alpha-cyclodextrin solution (125 mg/ml) to 240 μl of Bradford reagent. Orange: 10 μl of BSA dilutions in SDS buffer, reagent made by adding 20 μl alpha-cyclodextrin solution (125 mg/ml) to 230 μl of Bradford reagent. Brown: 10 μl of BSA dilutions in SDS buffer, reagent made by adding 20 μl alpha-cyclodextrin solution (125 mg/ml) to 105 μl of water and 125 μl of 2× concentrated Bradford reagent. Blue: 5 μl of BSA dilutions in RIPA buffer, reagent made by adding 20 μl alpha-cyclodextrin solution (125 mg/ml) to 230 μl of Bradford reagent. Light blue: 10 μl of BSA dilutions in RIPA buffer, reagent made by adding 40 μl alpha-cyclodextrin solution (125 mg/ml) to 85 μl of water and 125 μl of 2× concentrated Bradford reagent. This special reagent was tried to normalize the response of the assay to 10μl RIPA buffer. In panel A, the abscissae values are the concentration of BSA (in mg/ml) of the sample, which can be of 5 or 10μl size depending on the assay conditions. In panel B, the abscissae values are the amount of BSA per well.
Table 3.
False blind experiments for assessing the accuracy of the assay.
Fig 7.
SDS electrophoresis experiment made with various detergent containing extraction solutions.
RAW264 cells were extracted under various conditions (details reported in Material and methods). Urea: 7 M urea, 2 M thiourea, 4% CHAPS. SDS: 2% SDS, 5% mercaptoethanol. RIPA: 1% Igepal, 0.5% deoxycholate, 0.1% SDS. Native: 0.1% SB 3–14.
Table 4.
Flowchart for protein assay in the cuvette format.
Table 5.
Flowchart for protein assay in the microplate format.
For samples in Laemmli buffer, the scale of the assay (5 or 10 μl sample) must be determined first. The only practicable scale for samples in RIPA buffer is 5 μl of sample.