Protein Protocols Protein Protocols

28 Akins and Tuan
protein concentration falls within the desired range (see Note 4). Samples should be
either filtered or centrifuged to remove any debris prior to protein determination.
3.2. Selection of Microwave Oven
Microwave ovens differ substantially in their suitability for these assays. Desirable
attributes include fine control of irradiation time, a chamber size large enough to easily
accommodate the desired number of samples, and a configuration that results in a
homogenous field of irradiation so that all samples within the central volume of the
oven receive a uniform microwave dose (see Note 4). Samples should be placed in a
nonmetallic test tube rack in the center of the oven. A volume of room temperature
water is included in the oven chamber as well so that the total amount of fluid (samples
+ additional water) is constant from one assay to another (see Note 5).
3.3. Sample Irradiation
Once the samples are placed into the center of the microwave chamber, close the
door and irradiate the samples. Using the DC Protein Assay , a 10-s irradiation was
optimal as a replacement for the standard 15–min incubation. Using the BCA* Protein
Assay, a 20-s irradiation has proved adequate in most situations. We have found it most
convenient to use the highest setting on the microwave oven and to control exposure
using an accurate timer.
3.4. Reading and Interpreting Assay Results
After irradiation, the absorbance of each sample and standard should be determined
spectrophotometrically. Samples from Lowry assays should be read at 750 nm and
samples from BCA assays should be read at 562 nm. A standard curve of absorbance
values as a function of standard protein concentration can be generated easily and used
to determine the protein levels in the unknown samples. It is recommended that standard
curves be generated along with each assay to avoid any difficulties that may arise
from differences in reagents or alterations in total microwave exposure.
3.5. Summary
In summary, the microwave BCA protein assay protocol is as follows:
1. Combine samples and BSA standards with BCA assay reagent in polystyrene tubes;
2. Place samples into an all plastic test tube rack in the center of a microwave oven along
with a beaker containing a volume of room temperature water sufficient to make the total
volume of liquid in the chamber 100 mL;
3. Irradiate samples for 20 s on the highest microwave setting; and
4. Measure A562 for each sample and determine protein concentrations based on a BSA calibration
curve.
The microwave Lowry assay protocol is virtually identical:
1. Combine samples and Lowry assay reagents in polystyrene tubes;
2. Place samples into an all plastic test tube rack in the center of a microwave oven along
with a beaker containing a volume of room temperature water sufficient to make the total
volume of liquid in the chamber 100 mL;
3. Irradiate samples for 10 s on the highest microwave setting; and
4. Measure A750 for each sample and determine protein concentrations based on a BSA calibration
curve.