Protein Protocols Protein Protocols

38 Boerner et al.
2.2. Nitric Acid Protein Determination Method
1. SDS sample buffer: 2% (w/v) SDS; 4 M urea (prepared as an 8 M stock and deionized over
Bio-Rad AG501-X8 mixed bed resin prior to use); 62.5 mM Tris-HCl, pH 6.8 at 20–22°C,
and 1 mM EDTA with or without 5% (v/v) β-mercaptoethanol.
2. ACS reagent grade concentrated (70%) nitric acid.
3. BSA.
3. Methods
3.1. Nitric acid Protein Determination Method
The following is the recommended procedure for determining protein content in
samples solubilized in SDS sample buffer (see Notes 1 and 2):
1. Remove an aliquot containing 5–100 µg of protein. Bring sample volume to 10 µL.
2. Add 140 µL 70% nitric acid.
3. Incubate at 20–22°C for 2 h (see Note 3).
4. Utilizing suitable microcuvettes, measure absorbance at 358 nm with H 2O as a blank
(see Note 4).
5. Compare results to a standard curve constructed with varying amounts of BSA in 10 µL
SDS sample buffer subjected to the same conditions (see Notes 5 and 6).
3.2. Nitric Acid Protein Determination Method Modification One:
Whole Cells
Simple modifications allowed this procedure to be applied to other settings. When
protein content in whole cells is to be determined:
1. Wash cells with serum-free buffer (see Note 1).
2. Solublize cells directly in 70% nitric acid.
3. Incubate at 22°C for 24 h (see Note 3).
4. Determine absorbance at 358 nm (dilution in 70% nitric acid may be necessary; see Note 4).
5. Compare results to a standard curve constructed with varying amounts of BSA subjected
to the same assay conditions (see Notes 5 and 6).
3.3. Nitric Acid Protein Determination Method Modification Two:
Perchloric or Trichloric Acid Precipitates
When perchloric acid or trichloric acetic acid precipitates are assayed for protein
content (see Note 1):
1. Solubilize precipitates directly in nitric acid.
2. Incubate for 2 h at 22°C (see Note 3).
3. Determine absorbance at 358 nm (dilution in 70% nitric acid may be necessary; see Note 4).
4. Compare results to a standard curve constructed with varying amounts of BSA subjected
to the same assay conditions (see Notes 5 and 6).
4. Notes
1. Prior to assay for protein, cells must be washed in serum-free medium or physiological
buffer to remove (exogenous) protein and cross-reacting substances contained in culture
media.
2. In contrast to existing assays, the nitric acid method is not adversely affected by urea,
reducing agents, and most nonionic and ionic detergents. As a consequence, it is possible
to solubilize samples directly in SDS sample buffer, remove an aliquot for protein deter-