A highly sensitive, colorimetric protein assay that overcomes interference of common laboratory agents present in protein solutions and shows minimal protein-to-protein variation.
The assay is unaffected by the presence of common laboratory agents, such as reducing agents, chelating agents, detergents, amines, sugars, chaotropes, salts, drugs, antibiotics, cobalt and other common laboratory agents (see tables 1 and 2). The NI™ Protein Assay is composed of two simple steps:
Universal Protein Precipitating Agent (UPPA™) is added to the protein solutions to rapidly precipitate total protein. Protein is immobilized by centrifugation and interfering agents in the supernatant are discarded. Protein concentration is assayed by mixing with an alkaline solution containing a known concentration of copper salt; the copper ions bind to the peptide backbone and the assay measures the unbound copper ions (see figure 2). The assay is independent of protein side chains minimizing protein-to-protein variation (see figure 3). The color density is inversely proportional to the amount of protein. The assay is supplied with a traditional bovine serum albumin (BSA) protein standard or a non animal protein standard.
Table 1: A selection of compounds and the maximum concentrations that are compatible with the NI™ Protein Assay.| Compound | Concentration | Compound | Concentration |
| Ammonium sulfate | 40% | N-Octyl glucoside | 0.5% |
| Brij® 35 | 1% | Phosphate buffer | 0.2M |
| CHAPS | 1% | Sarcosyl | 1% |
| CHAPSO | 1% | Sodium azide | 0.1M |
| CTAB | 1M | Sodium dodecyl sulfate (SDS) | 1% |
| Digitonin | 0.3% | Sucrose | 30% |
| DTT | 10mM | TCEP | 15mM |
| EDTA | 10mM | Thesit® | 2% |
| Glycerol | 30% | Thiourea | 2M |
| Guanidine.HCl | 6M | Tris | 0.2M |
| Guanidine thiocyanate | 6M | Triton® X-100 | 3% |
| HEPES | 0.1M | Triton® X-114 | 1% |
| Iodoacetamide | 15mM | Tween® 20 | 2% |
| 2-mercaptoethanol | 0.5% | Urea | 8M |
Table 2: NI™ Protein Assay is compatible with strong chaotropic extraction buffers| Buffer composition | Buffer composition |
| 4M urea, 1% SDS, 10mM EDTA, 0.8% 2-mercaptoethanol | 1% Sarcosyl, 0.8% 2-mercaptoethanol, 4M guanidine thiocyanate, 10mM EDTA |
| 6M urea, 2M thiourea, 4% CHAPS | 6M urea, 2M thiourea, 2% CHAPS, 2% ND SB 201 |
| 6M urea, 2M thiourea, 4% Nonidet® P-40 | 6M urea, 2M thiourea, 2% CHAPS, 2% SB 2 10 |
Features- Linear response 0.5µg-50µg protein
- Small sample requirement, only 1-50µl
- Unaffected by non-protein chemicals and agents
- Protocol time: ~30 minutes
- Long Shelf Life
Applications
- Estimate protein during protein purification, electrophoresis, cell biology, molecular biology, and other research applications.
- Suitable for protein samples containing common laboratory agents, such as reducing agents
(ß-mercaptoethanol, dithiothreitol), chelating agents (EDTA), detergents, amines (Tris), sugars and many other agents. - Suitable for samples containing chaotropic agents such as urea, thiourea, guanidine hydrochloride, guanidine thiocyanate, ammonium sulfate, drugs, antibiotics, cobalt, and numerous other agents and extraction buffers.
- Suitable for determination of protein concentration in cellular fractions, tissue & cell lysates and chromatography purification fractions.
- Suitable for dilute protein solutions.