- 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
Posts Tagged ‘Proteomics’
Hercules, CA — March 13, 2012 — Bio-Rad Laboratories, Inc.’s PROTEAN i12 IEF system won a Laboratory Equipment Readers’ Choice Award in the Basic Lab Equipment category. Theaward will be presented today at a special reception during the Pittcon Conference & Expo 2012 being held at the Orange County Convention Center in Orlando, FL.
“The PROTEAN i12 IEF system individually controls each of its 12 lanes, allowing researchers to generate reproducible 2-D gels in less time and with total confidence,” said Renee Lemaire-Adkins, Bio-Rad Marketing Manager, Lab Separations Division. “It is a great honor to have our IEF technology recognized by the researchers and laboratory professionals who compose Laboratory Equipment’s readership.”
The 4th annual award celebrates “excellence in product design and performance for the tools and materials used by scientists and engineers in research laboratories.” Laboratory Equipment readers voted for products online.
The PROTEAN i12 IEF system is the industry’s only isoelectric focusing (IEF) system designed to simultaneously run up to 12 immobilized pH gradient (IPG) strips in 12 independently programmed lanes, which allows users to confidently run 12 different conditions at one time. Although many available IEF systems have multiple lanes, all but the PROTEAN i12 system depend on a single power supply, allowing only one set of conditions to be run at a time.
By independently controlling voltage and current levels in each lane, the PROTEAN i12 IEF cell allows users to optimize different sample and pH conditions and run samples from different experiments simultaneously.The independent lane control prevents lane-to-lane sample interference, resulting in faster, more accurate, reliable, and reproducible focusing.
Visit http://bit.ly/proteani12pr or Bio-Rad’s Pittcon Booth #1412 to learn more about the PROTEAN i12 IEF system. You can find more about the Laboratory Equipment Reader’s Choice Award at http://bit.ly/2012readerschoice.
Proteomics is about to take a big leap forward, that is if the NIH can help it.
Last week, the NIH put out a request for information aimed at determining how best to accelerate research in disruptive proteomics technologies. The organization is hoping that submissions will aim to greatly outperform current mass spec technologies and introduce an all new way of advancing proteomic questions.
According to the proposal:
The Disruptive Proteomics Technologies (DPT) Working Group of the NIH Common Fund wishes to identify gaps and opportunities in current technologies and methodologies related to proteome-wide measurements. For the purposes of this RFI, “disruptive” is defined as very rapid, very significant gains, similar to the “disruptive” technology development that occurred in DNA sequencing technology.
These are exciting times for the field of proteomics. Don’t be left behind! Click here to find out more on how to get involved today!
Interesting story out of the Biophysical Society’s 56th Annual Meeting.
Several neurodegenerative diseases – including Alzheimer’s and ALS (Lou Gehrig’s disease) – are caused when the body’s own proteins fold incorrectly, recruit and convert healthy proteins to the misfolded form, and aggregate in large clumps that gum up the works of the nervous system. Now scientists have developed an algorithm that can predict which regions of a protein are prone to exposure upon misfolding, and how mutations in the protein and changes in the cellular environment might affect the stability of these vulnerable regions. These predictions help scientists gain a better understanding of protein dynamics, and may one day help in developing treatments to effectively combat currently incurable neurodegenerative diseases.
The algorithm uses the energy equations of thermodynamics to calculate the likelihood that certain stretches of protein will be displayed when the protein misfolds. Since the exposed regions are specific to the misfolded version of the protein, researchers can use these regions as targets for diagnostic and therapeutic treatments. The algorithm can be adapted for different proteins and predicts several potential target regions for each protein. The group has used it to study neurodegenerative disease-causing proteins as well as misfolded proteins that have been implicated in some cancers.
Yesterday we told you about how to get more data from your western blots by utilizing multiplex fluorescent detection. Today, we will provide you with a primer on fluorescent detection taken from the Bio-Rad Laboratories Protein Blotting Guide.
In fluorescence, a high-energy photon (ℎVex) excites a fluorophore, causing it to leave the ground state (S0) and enter a higher energy state (S’1). Some of this energy dissipates, allowing the fluorophore to enter a relaxed excited state (S1). A photon of light is emitted (ℎVem), returning the fluorophore to the ground state. The emitted photon is of a lower energy
(longer wavelength) due to the dissipation of energy while in the excited state.
When using fluorescence detection, consider the following optical characteristics of the fluorophores to optimize the signal:
- Quantum yield — efficiency of photon emission after absorption of a photon. Processes that return the fluorophore to the ground state but do not result in the emission of a fluorescence photon lower the quantum yield.Fluorop hores with higher quantum yields are generally brighter
- Extinction coefficient — measure of how well a fluorophore absorbs light at a specific wavelength. Since absorbance depends on path length and concentration (Beer’s Law), the extinction coefficient is usually expressed in cm–1 M–1. As with quantum yield, fluorophores with higher extinction coefficients are usually brighter
- Stokes shift — difference in the maximum excitation and emission wavelengths of a fluorophore. Since some energy is dissipated while the fluorophore is in the excited state, emitted photons are of lower energy (longer wavelength) than the light used for excitation. Larger Stokes shifts minimize overlap between the excitation and emission wavelengths, increasing the detected signal
- Excitation and emission spectra — excitation spectra are plots of the fluorescence intensity of a fluorophore over the range of excitation wavelengths; emission spectra show the emission wavelengths of the fluorescing molecule. Choose fluorophores that can be excited by the light source in the imager and that have emission spectra that can be captured by the instrument. When performing multiplex western blots, choose fluorophores with minimally overlapping spectra to avoid channel crosstalk