Posts tagged ‘Proteomics’
One of the central questions in human biology is to understand how our genes determine which diseases we get and how severe they might be. Knowing just the DNA sequence, or the blueprint, is not enough. We must figure out how proteins, the genes’ products, work too.
Now an international team of researchers, jointly led by Dr. Fritz Roth (at Mount Sinai Hospital’s Lunenfeld-Tanenbaum Research Institute and the Donnelly Centre of the University of Toronto), and Dr. Marc Vidal (with the Dana-Farber Cancer Institute and Harvard Medical School in Boston), have produced the largest ever map of human protein interactions. This publicly available resource will be invaluable to anyone trying to understand complex genetic traits and develop new disease therapies.
Understanding the checks and balances that govern when and how much cells grow is key to understanding cancer. A study published in November 2013 by the Gingras lab uncovers pieces of the complex mosaic of molecular interactions or signals that govern the normal growth of cells, tissue, and organs.
Within all animal cells an important series of switches causes them to stop growing once a tissue has attained the right size. “This system is called the Hippo pathway because deregulation of this system leads to overgrowth, a ‘hippopotamus’ phenotype. The Hippo pathway consists of proteins that interact with one another, sense other control systems within our cells, and send signals to stop the cell growth,” says Dr. Gingras.
“Our study identified 749 interactions between proteins and enzymes that play a role in telling a cell when to stop growing. Of these, 600 have not been previously recognized in the Hippo pathway,” she says.
“These findings are promising because, to date, there are no drugs directed at the components of the Hippo pathway,” adds Dr. Jim Woodgett, Director of the Lunenfeld-Tanenbaum Research Institute. “Anne-Claude’s team’s work has added many new candidates for therapeutic intervention that may, for example, help in restricting the uncontrolled growth of tumour cells.”
- Blocking was incomplete
- Increase the concentration of blocker
- Increase the duration of the blocking step
- Use a different blocking agent
- Use a pure protein such as BSA or casein as a blocker
- Increase the number, duration, or stringency of the washes
- Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween 20
- Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding
- Remove the blot from the substrate solution when the signal-to-noise level is acceptable, and immerse in diH2O
- Discard and prepare fresh gels and transfer solutions
- Replace or thoroughly clean contaminated foam pads if a tank blotter was used
- Reduce the amount of protein on the gel or SDS in the transfer buffer
- Add a second sheet of membrane to bind excess protein
- Increase antibody dilutions
- Perform a dot-blot experiment to optimize working antibody concentration
- Clean the trays or use disposable trays
Which camera is best for chemiluminescent detection?
Four Great Tips for Effective Protein Blotting
Test your knowledge of protein blotting membranes
Efficient Electrophoresis and Protein Blotting: Aiding Advances in Cardiomyopathy Research
Protein blotting guide for novice and advanced users
Western blotting, the backbone of protein research, is a universal lab procedure. While the premise of a western blot is simple — target proteins are identified and quantitated via antibody-antigen interactions — the traditional workflow is labor intensive and time consuming. Researchers have long sought a faster solution — an archetype that would streamline the entire process of separation, transfer, and visualization of results without compromising data quality.
For decades, a lucky subset of molecular biologists have been engaged in the study of beer diligently trying to figure out the science behind the drink’s clarity, taste and foam content. While most of us are content with studying beer after work hours, researchers from France and Japan worked many hours to uncover 18 proteins within the beer proteome that likely influence its delectable characteristics. Although their results are certainly noteworthy, the protocols utilized suffered from sample input limitations (less than 10 ml of sample per run) and consequently a relatively small number of proteins were discovered. Then came the Italians…
In a paper published in the Journal of Proteome Research , scientists from Milan utilized a protein enrichment strategy to identify 54 types of proteins from Italian-bottled Splügen beer. The group used Proteominer technology from Bio-Rad Laboratories to magnify low-abundant proteins that would have otherwise been lost with a protein depletion approach. They then analyzed the remaining fractions by mass spectrometry analysis and identified over 40 proteins that were present in trace amounts in the beer sample.
Lead author Pier Giorgio Righetti, of the The Polytechnic Institute of Milan told Discovery News “This opens up a completely new horizon in beer analysis in general, and also in the analysis of any beverage. We are now analyzing a lot of other beverages and finding a lot of surprising things that producers don’t know are in their beverages.”
Power supplies that are used for electrophoresis hold one parameter constant (either voltage, current, or power). The PowerPac™ HC and PowerPac Universal power supplies also have an automatic crossover capability that allows the power supply to switch over to a variable parameter if a set output limit is reached. This helps prevent damage to the transfer cell.
During transfer, if the resistance in the system decreases as a result of Joule heating, the consequences are different and depend on which parameter is held constant.
Transfers Under Constant Voltage
If the voltage is held constant throughout a transfer, the current in most transfer systems increases as the resistance drops due to heating (the exception is most semi-dry systems, where current actually drops as a result of buffer depletion). Therefore, the overall power increases during transfer, and more heating occurs. Despite the increased risk of heating, a constant voltage ensures that field strength remains constant, providing the most efficient transfer possible for tank blotting methods. Use of the cooling elements available with the various tank blotting systems should prevent problems with heating.
Transfers Under Constant Current
If the current is held constant during a run, a decrease in resistance results in a decrease in voltage and power over time. Though heating is minimized, proteins are transferred more slowly due to decreased field strength.
Transfers Under Constant Power
If the power is held constant during a transfer, changes in resistance result in increases in current, but to a lesser degree than when voltage is held constant. Constant power is an alternative to constant current for regulating heat production during transfer.