Protein concentrations have traditionally been determined by the method of Lowry and co-workers (Lowry et al., 1951) using the Folin-Ciocalteau reagent, but this method has been found to be interfered with by salts or detergents in the sample. However, the presence of salts or detergents etc. in the sample interferes with this method, and Peterson (1977) describes an improved method which partially solves these problems. This experiment was derived from the Laboratory Guide for Protein Purification and Identification by Hau-Chu Chu.
Operation method
Measurement of protein concentration in the presence of interfering substances
Materials and Instruments
Reagent A Reagent B Bovine serum albumin Sodium deoxycholate Sodium trichloroacetate Move Materials For more product details, please visit Aladdin Scientific website.
Bovine serum albumin (BSA)
Sodium deoxycholate (0.15%)
Sodium trichloroacetate (TCA; 72%)
Reagents
Reagent A
Reagent B
(For the recipe, see Preparation of reagents pp.234-240)
Procedures
1) Prepare lmg/ml BSA solution in distilled water, dispense it, and freeze it at -20°C for later use.
2) Prepare a standard curve, and prepare the following mixtures in 1.5-ml microcentrifuge tubes: 3) Dilute membrane protein samples (1:10) with distilled water, and take 5-ul, 10-ul, and 20-ul aliquots for determination.
3) Dilute the membrane protein samples (1:10) with distilled water and take 5-ul, 10-ul, 20-ul aliquots for determination. For solubilized membrane protein or WGA column chromatographic fraction, no dilution is necessary, 10ul can be taken. Take another 10ul of buffer in the blank control tube to determine the absorption value of the buffer component.
Note: Steps 4-8 can be omitted if there are no interfering substances, their contents are negligible, or they are properly controlled. In such cases, dilute each sample to 0.4 ml with distilled water.
4) Dilute membrane protein sample to 1 ml with distilled water.
5) Add 0.1 ml of 0.15% sodium deoxycholate to each tube. Mix and allow to stand at room temperature for 10 min.
6) Add 0.1 ml 72% TCA to each tube. Mix and centrifuge in a microcentrifuge for 15 min at room temperature.
7) Discard supernatant. Can be pipetted efficiently.
8) Add 0.4 ml distilled water to each tube.
9) Add 0.4 ml Reagent A to each tube, mix well with shaking and leave at room temperature for 10 min.
10) Add 0.2 ml Reagent B to each tube and mix immediately. Incubate for 30 min at room temperature.
11) Read absorbance at 750nm.
Note: Read absorbance within 2 hours unless standard protein samples are included in each sample set. This method can be easily converted to a micro-assay by reducing the volume of the 8-10 steps by a factor of 10 to increase the sensitivity of the 1%~2% per hour coloration at room temperature. Steps 1 to 7 are the same as for the macronutrient assay, except that the amount of protein used in the standard curve is reduced to 0.1-10ug.
12) Plot the amount of protein in the standard sample tube against the absorbance at 750nm and draw a standard curve.
13) Based on the absorbance at 750nm of each sample, the amount of protein in each sample will be calculated from the standard curve.