Many biological and medical laboratories utilize a technique known as "western blotting" to detect proteins, such as from cell extracts or blood serum samples. The technique essentially takes a small drop of protein solution, and separates it into a long band of proteins.
The position of a protein within the band is based on a number of characteristics (such as protein charge or shape), and a given protein will always end up at the same position within the band under the same separation conditions. Thus, observing a spot at a given point within the band tells one that a specific protein is present within the sample.
Western blotting is a common technique, for example to diagnose HIV. If antibodies (proteins designed by the body to combat bacteria and viruses) to HIV are detected within a protein band, the patient in question has HIV.
The technique is popular because it is cheap and reliable. However, improvements in detection efficacy, enabling very small sample volumes to be utilized and very small protein quantities to be detected, would enhance its utility even further.
Tania Vu (Oregon Health and Science University) and coworkers have made such improvements. They have utilized fluorescent nanoparticles to improve the detection sensitivity of western blotting by a factor of 1000, and reduce the required sample volume by a factor of 100.
Unique advantages of the fluorescent nanoparticles.
The fluorescent nanoparticles the scientists chose to improve the detection efficacy of western blotting are known as quantum dots. They have many advantages over conventional fluorescent small molecules.
They are bright and are not easily destroyed by shining light on them. Additionally, one color of light can activate nanoparticles designed to emit different colors of fluorescence (which is important for quantitation purposes, but will not be elaborated upon here).
The scientists designed the nanoparticles to chemically bind to specific proteins. Thus, if fluorescence is observed at a given spot within a protein band created via western blotting, the protein of interest is there.
Furthermore, the intensity of fluorescence is related to the number of proteins at that given location. This tells one the concentration of a given protein in the sample (which may, for example, be related to whether or not a patient has cancer).
Visualizing the protein bands.
A challenge is that the scientists wanted to visualize their protein bands with a fluorescence microscope. However, the media on which the protein bands are present (polyvinylidene fluoride) is not fully transparent.
They needed to develop a method to make them transparent to visible light without washing away the protein bands. Their approach was to penetrate the media with polydimethylsiloxane, imparting transparency while also enhancing the stability of the protein bands.
Improved detection sensitivity.
One of the scientists' primary goals in this research was to improve the detection sensitivity of western blot analysis, i.e., detect even smaller amounts of protein in a sample. This is of medical relevance, because sometimes only a small amount of a disease-indicative protein is present in a blood sample.
The scientists were able to quantitate 10 picograms of nerve growth factor, a protein important for the maintenance and survival of nerve cells. This is improved by a factor of 1000 over the 10 nanograms possible with conventional western blot analysis.
The same enhanced efficacy level was observed with Factor XI, a protein important in blood clot formation and which is only present in small amounts in blood serum. It is clear that the scientists' technique improves the detection sensitivity of western blot analysis.
Reducing the required sample volume.
Another important goal of this research was to reduce the sample volume required for western blot analysis. This is of biochemical relevance, such as for experiments aimed at elucidating the protein composition of a small subgroup of cells within a large population.
Due to the enhanced detection sensitivity of the scientists' protocol, they were able to detect tyrosine kinase receptor A (a protein which regulates the conversion of generic nerve cells into specialied nerve cells) from a sample containing only 100 cells. There was only 50 nanograms of protein in these cells.
In contrast, 100,000 cells (possessing 50 micrograms of protein) is required for a conventional western blot analysis. This means that these scientists' approach is improved by a factor of 1000.
Similar results were observed for actin, a protein important for cell shape and motility, and for CrkL, a protein critical in the progression of a chronic bone cancer. It is clear that the scientists' technique reduces the sample volume required for western blot analysis.
Further optimization.
The scientists' technique is based on detecting "individual" nanoparticles (approximately 75% of the visualized fluorescence spots were comprised of nine or fewer nanoparticles). Consequently, it is especially important to correct for movement in and out of the plane of view (which impacts measured fluorescence intensity, and therefore protein quantitation) when imaging along the length of a protein band.
The chemical binding efficacy of the nanoparticles could be improved to further enhance protein detection efficacy. Even though the nanoparticles are comprised of rare metals and are both expensive and challenging to prepare, this detection protocol should still be of wide utility towards medical diagnostics screening and other biochemical analyses.
for more information:
Scholl, B., Liu, H. Y., Long, B. R., McCarty, O. J. T., O'Hare, T., Druker, B. J., & Vu, T. Q. (2009). Single Particle Quantum Dot Imaging Achieves Ultrasensitive Detection Capabilities for Western Immunoblot Analysis ACS Nano, 3 (6), 1318-1328 DOI: 10.1021/nn9000353