Enhancing Detection of Low-Abundance Proteins

Low-abundance proteins: how to enhance detection and quantification?

Western blotting visualises numerous molecular events. Some of them include expression and localisation of proteins, post-translational modifications and protein-protein interactions.

The main issue with illuminating such events is that often they are very subtle, leading to researchers having a hard time detecting proteins with low abundance. We have decided to share some advice on how to perform several steps that can ensure higher quality of the Western blot.

Preparing the samples:

The first improvements can be made at the initial sample preparation step. Several methods can optimise protein recovery from the sample.

Lysis buffers:

During lysis, the cell membrane is broken and the cellular components are re-suspended in a soluble form. Ionic or non-ionic detergents are the main active agent in this step, with SDS being the most common ionic and Triton-x and NP-40 being the best-known non-ionic detergents.

In general, ionic detergents are considered to be harsher, because they can disrupt the structure of proteins. Because of this quality, they are recommended for use with antibodies, raised against denatured forms or synthetic proteins of the protein of interest.

On the other hand, non-ionic detergents are better suited for primary antibodies against native proteins, because they induce less denaturation. Knowing which lysis buffer to use can greatly improve the quality of the experiment.

Ways of determining the concentration of the recovered protein:

This step can be done in three different ways. All of them use colourimetric assays, able to utilize set concentrations of BSA, in order to create a standard curve for sample evaluation. The assays include Lowry, BCA and Bradford assay.

Gel electrophoresis:

Choosing which gel to use is easier when you know the size of the protein of interest. Using a high percentage gel for a low MW protein and vice versa is advisable for optimal resolution.

Transfer times:

Larger proteins usually take longer to transfer compared to smaller ones. Choosing which transfer protocol to use greatly relies on the size of the protein of interest. If it is on the larger side, you can extend the time as long as the protocol recommends, but if you leave it for any longer than that, the protein can be lost.

Furthermore, choosing the right membrane is also important for the transfer. In the case of low abundant proteins, PVDF is more suitable, because it has greater binding capacity.

Blocking and washing:

The process of blocking is responsible for eliminating substantial amounts of non-specific binding. Usually, BSA or re-suspended skimmed milk are used for blocking this type of binding to the membrane.

These blocking agents are suspended either in Tris-Saline including detergent. If detecting a low abundant protein is required, the concentration of the blocking agent has to be reduced.