General Lab Considerations

• Use clean glassware and supplies
• Disposable cuvettes are an option
• Make sure cuvettes are clean of all residues
• Protein assays are strongly influenced by the composition of the proteins present in your sample
• Become familiar with spectrophotometry before proceeding
• Always let a spectrophotometer warm up for 15-20 minutes before using
• Know the limits of the spectrophotometer with which you are using

Make sure all glassware is clean.  It is difficult to get accurate results if your supplies are dirty.  High quality detergents and distilled water should be used to clean equipment.  In some cases, such as the Bradford assay, it may be more convenient to use disposable cuvettes.  Some of the described assays will leave residues on cuvettes.  If you are having trouble removing residues from cuvettes, try cleaning with 50% nitric acid (use caution!).  Also, a wash with ethanol or methanol often works well to remove contaminating proteins.  A thorough wash with distilled water between determinations is always a must.

Before starting a particular analysis, it is highly recommended that you review the literature of your field to ascertain the standard methods of protein determination that are being used.  This in no way means that you have to use this particular method, it is just a good idea to be aware what other researchers in you field are utilizing to determine protein concentration.

One thing to keep in mind throughout the discussion of the methods is that protein assays only detect the presence of certain parts of proteins.  The abundance and presence of these parts (such as particular amino acids) is highly variable from one protein to another and will have a big influence on the choice and methodology of the assay.

All of the described methods rely on the use of a spectrophotometer.  Before beginning these assays you should make yourself familiar with the operation and use of a spectrophotometer.  Note that spectrophotometers take approximately 15-20 minutes to warm up before the wavelength of the lamp becomes stable.  You should also take note of the limitations of the specific spectrophotometer that you are using to conduct the assay.  Take note especially of the upper limit of your absorbance reading on your spectrophotometer.  You need to make sure that both your standard curve and your unknown determination fall within the absorbance limits of your spectrophotometer.  Most spectrophotometers cannot linearly read absorbance values beyond 2.0 units.  Thus, you may have to dilute your protein of interest to work within this range.