A team of New York Stem Cell Foundation (NYSCF) Research Institute scientists led by David Kahler, PhD, NYSCF Director of Laboratory Automation, have developed a new way to generate induced pluripotent stem (iPS) cell lines from human fibroblasts, acquired from both healthy and diseased donors. Reported in PLOS ONE, this cell-sorting method consistently selects the highest quality, standardized iPS cells, representing a major step forward for drug discovery and the development of cell therapies.
Posts Tagged ‘research methodology’
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.
- Blocking was incomplete
- Increase the concentration of blocker
- Increase the duration of the blocking step
- Use a different blocking agent
- Blocker was impure
- Use a pure protein such as BSA or casein as a blocker
- Wash protocols were insufficient
- 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
- The blot was left in the enzyme substrate too long (colorimetric detection)
- Remove the blot from the substrate solution when the signal-to-noise level is acceptable, and immerse in diH2O
- Contamination occurred during electrophoresis or transfer
- Discard and prepare fresh gels and transfer solutions
- Replace or thoroughly clean contaminated foam pads if a tank blotter was used
- Excessive amounts of protein were loadedon the gel or too much SDS was used inthe transfer buffer. Proteins can pass through the membrane without binding and recirculate through a tank blotting system.
- Reduce the amount of protein on the gel or SDS in the transfer buffer
- Add a second sheet of membrane to bind excess protein
- The primary or secondary antibody was too concentrated
- Increase antibody dilutions
- Perform a dot-blot experiment to optimize working antibody concentration
- Incubation trays were contaminated
- Clean the trays or use disposable trays
I just saw the following news release out of the American Academy of Pediatrics and I am not sure what to make of it. From what I see, the study has an n of 10 and tries to pull “accurate” information from a group of fathers who were interviewed in a focus group setting.
I know that this is preliminary data out of an AAP presentation and that my critisism may be unjustified. Nonetheless have a look at the press release and let me know your thoughts.