Question

What are the controls in total protein assay? Why do you need these control samples?

Why do you need to dilute the unknown samples to three different dilutions? How do you make 1:10, 1:100, and 1:1000 dilutions in the experiment?

Answer 1

Protein concentration quantitation is an integral part of any laboratory workflow involving protein extraction, purification, labeling or analysis. Cell lysates are assayed to measure the protein yield from the lysis step and to normalize multiple samples for downstream application or for side-by-side comparison. Proteins obtained from a purification procedure are assayed to determine yield.Purified proteins that will be labeled with biotin or conjugated to reporter enzymes are typically assayed to ensure that the labeling reaction is prepared with appropriate stoichiometry. Given the wide range of reagent components that may be present in different kinds of samples, it is amazing that there exist protein assay reagents that are capable of reliably and specifically measuring the protein concentration.

Important criteria for choosing an assay include:

• Compatibility with the sample type and components
• Assay range and required sample volume
• Protein-to-protein uniformity (see below)
• Speed and convenience for the number of samples to be testedAvailability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay.

Inaccuracy resulting from a small amount of interfering substance can be eliminated by preparing the protein standard in the same buffer as the protein being assayed. For higher, incompatible levels of interfering substances, other strategies are necessary:

• Choose a different protein assay method or a version of the same assay method that includes components to overcome the interference.
• Dialyze or desalt the sample to remove interfering substances that are small (i.e., less than 1000 daltons), such as reducing agents.Precipitate the protein in TCA or other appropriate reagent, remove the solution containing the interfering component, and then re-dissolve the protein for analysis. This illustration provides an overview of how protein dialysis methods are used to remove substances that may contaminate protein samples and interfere with downstream applications.