The invention of Western blotting appeared 30 years later, this technology is still reliable identification of specific proteins to obtain the key. Many recent surge of products using a variety of methods to improve the repeatability of the Western blot experiments, sensitivity, quantitative as well as speed.
Three people have been thought that the development of a protein immunoblotting method, but only a human can be regarded as “Western Blotting (Western blot),” the name, he was at the time 西雅图哈钦森 Cancer Research Center Bob Nowinski laboratory work The W. Neal Burnette. “Western Blotting” implicit in the naming of the Southern Blotting (Edwin Southern in 1975 invented a technique that uses plastic, nylon membrane and absorbent paper to identify an individual in the complex specific DNA sequences), Northern Blotting (subsequently invented the Similar strategies for the identification of RNA) and West Coast (West Coast) of Nowinski laboratory three intended.
Burnette’s technical achievements was to be published until 1981. He retrospect, when reviewers “special” against the use of “Western blotting” the name. Nevertheless, this name is followed down, and western blot technology has become the most widely used one of immunochemical techniques.
Western blotting The first step involves using gel electrophoresis (Gelelectrophoresis) protein separation according to size, and then through the membrane in the gel, the proteins were transferred to a membrane (usually a nitrocellulose membrane or a polyvinylidene difluoride membrane ), and in the film, plus a number of films absorbent paper, then layer on this heap buffer solution by capillary action so that you can pull up the protein dragged membrane. This is the so-called wet or trough transfer method.
The other two techniques are dry and semi-dry transfer method, the two methods than the traditional wet transfer method is faster and more regular, but for high molecular weight proteins, the lower the efficiency. For semi-dry transfer method, the film and the adhesive is placed on a filter paper soaked buffer layer, these are sandwiched between the cathode and the anode, the current can be driven proteins were transferred to the membrane.Three methods, the dry transfer fastest, but the transfer efficiency is the lowest.
After transfer, the membrane is placed in a solution diluted to a protein non-specific protein binding closed. The membrane was then incubated with an anti-elution, and then the signal is detected with the labeled probe is incubated with secondary antibody.
The final step is to test, usually fluorescence or chemiluminescence method. Chemiluminescence detection, and the enzyme cross-linked secondary antibodies capable of detecting the antigen react to produce light, the film or the image forming apparatus can be captured. In fluorescence detection, the antibody labeled probe is fluorescent Group.
The main advantage of the fluorescence detection method is that it can detect more protein, and the signal is more consistent. Therefore, compared with chemiluminescence detection, but also more conducive to quantitative research.
Many Western blot related manufacturing equipment, software and consumables company is trying to promote some or all of the steps of the automation of this experiment can be expected to become more simple and effective. In the experiment, while adding some validation points, enabling researchers to experimental progress can be closely monitored, and even re-create the entire process. Scientists are looking for is efficiency, robustness and strategies, and then to be able to help them avoid wasting precious very expensive antibodies.
Accelerate the process of immunodetection
Transfer, antibody incubation, the sample and elution step occupied Western blotting experiments 80% of the time, EMD Millipore company “Western blotting Solutions’ product manager Michele Hatler this introduction.
This California-based company launched Timex Kula SNAP id 2.0 protein detection system to speed up the whole process, using a vacuum device pushed through the membrane reagent, rather than rely on diffusion. This allows time for the immunoassay shortened from four hours to 30 minutes, Hatler representation. She explained: “We are truly committed to improving workflow efficiency western blotting, we still follow the traditional steps away, just added a vacuum.”
Hatler that version 2.0 was launched in September 2012, and compared to the previous version has several advantages. It can use a gel medium size (8.5 × 13.5 cm) and mini-gel (7.5 × 8.4 cm) and can only be used before the mini-gel.
“This equipment is very cheap, and the operation is very simple, but effectively improve the efficiency of the experiment, saving experimental time.” Hatler said.
Bio-Rad (Bio-Rad) the Trans-Blot Turbo transfer system is the experimental protein countertops sized instrument, able to provide fast and efficient transfer. Thanks to a new transfer buffer formulations, special filter material and enhanced current intensity (by an integrated power regulator), the system can be as short as three minutes to complete the transfer, and the slot blot results can be style shift comparable. The traditional semi-dry transfer system requires 15-60 minutes time, and often do not provide the high molecular weight proteins were transferred powerful results.
Add verification points to relieve anxiety
Western blotting of the high failure rate is often disheartening, especially if you need several days to return a result. “This is a very unstable technology,” Bole company “Western blot Groups” marketing manager Ryan Short said, “We are user survey found that half of the users report that they use this technique failure rate is at least 25 percent.”
Short added: “Almost no chance to check the experiment meets expectations. So there was the anxiety we are introducing the concept of visual verification points to increase the confidence and certainty.”
The company’s standard and Mini-PROTEAN Free precast gel staining, using its ChemiDoc MP gel imaging system allows researchers to quickly observe their protein is correctly loaded into the gel, and then confirm the transfer of high quality protein, easy to decide whether we should turn to the next step processes or start again.ChemiDoc MP system for chemiluminescence and fluorescence speckle imaging technology multivariate designed to use on personal computers ImageLab software to operate.
These verification point “really useful” in the laboratory using the system at UC Davis assistant professor Aldrin Gomes said: “We can be imaged these free stained gel, without adding extra dye, and can quickly determine run the sample on the gel is a problem, we’ll go after the protein transfer film on the gel imaging, if any one step problems, we can stop the experiment at this point the process. ”
Quantitative imaging and software to improve
While many scientists still using western blot analysis of protein film, the world face began to appear more and more wide range of free recording film gel imaging system, these systems can be used to image and analyze the chemiluminescent blot, dot blot, or both fluorescence detect both imprinting. Many companies have started to work, eager to manufacture and convenient as possible imager and analysis software. Many companies offer a touch-tone image capture system, and its size can be in the experimental stage operation.
Syngene Company in April 2012 launched a very simple one button operation system PXi, chemiluminescent and fluorescent Western blot analysis. The system is based on the company’s G: BOX imaging system, and is equipped with GeneSys imaging software.
October 2012, Thermo Fisher Corporation (Thermo Fisher Scientific) introduced myECL imager using ultraviolet and visible light transmission method, specialized filters and a charge-coupled device (CCD) camera technology to capture and analyze proteins and protein blotting and nucleic acid gels.
The sensitivity of the CCD camera twice higher than the X-ray film, according to the company, the instrument’s dynamic range of up to 10 times. Users simply click on one of the best touch screen preset buttons, without focusing or adjusting camera settings. Users can also set custom exposure, and can also set up five different exposure, or use the default exposure parameters. This imager instrument occupies very little space, can be used in the experimental stage.
Aplegen company Omega Lum G gel recording system also has an automatic focus feature, you can operate in the experimental stage, and equipped with a integrated tablet. UVP company in January 2012 launched a Chemi-Doc-It TS2 Imager, which feature an integrated computer and touch screen that allows the user to adjust exposure, aperture, zoom and focus parameters, when the user presses the live preview (Live Preview ) button, the images also provide examples.
LI-COR latest version infrared imaging system Odyssey CLx, the version number has more than six pairs of the dynamic range, and the former has only a version number of more than four pairs of the dynamic range. When the results of the Western blot analysis, the scope of this increase can eliminate saturation, providing a wider range of linear quantitative data can be converted.
“The researchers not only in their imprinting does not reach saturation, and they can also put multiple blot imager, need not worry as these blots different settings to adjust.” Nebraska in Lincoln City, senior product marketing Manager Jeff Harford explains.
Rehana Leak is America Duquesne University in Pittsburgh (Duquesne University), assistant professor, she uses a LI-COR Odyssey CLx system how cells adapt to low levels of stress research. “Odyssey Imager advantage is that it is a 16-bit imager, 700 and 800 nm and has two infrared wavelengths, one can image two spots.” Leak said, “This means that we can work together to detect phosphorylated and total conformation protein. “two protein isoforms by other methods difficult to distinguish synchronization because they are very similar groups.
Leak noted, Odyssey imager 216 to visualize infrared signal shadow, compared, X-ray film showing only 150 gray shadow. “This transition from 150 to 65,000 infrared shadow, so this method can obtain a higher resolution.” She continued, “This is an unusual quantified.”
December 2012, LI-COR announced a new digital imaging system, namely C-DiGit system that can be used for chemiluminescent blots. The system will be launched in 2013. “Like recent years digital cameras replace film cameras, we believe that C-DiGit chemiluminescence system will replace the film.” LI-COR senior scientist Jon Anderson says, “This technology can provide films of all users are accustomed to the sensitivity and image quality while significantly improve the quality of the digital image. ”
Software is always intuitive
Western blot analysis of traditional software requires the user to export a spreadsheet from the image data, and the subsequent homogenization by calculation and analysis done manually. BioRad in September 2012 released by Image Lab 4.1 software for housekeeping proteins or protein provides embedded total loading, automated homogenization.
“This is the best I have seen in the market for one of the products.” Gomes said he is studying the proteasome and troponin in cardiac and skeletal muscle tissue in rats. “The best thing is that it is completely free.” Image Lab 4.1 software than he previously used in the lab image analysis software should be simple, Gomes went on to say that almost everyone can grasp. “It really enhances our quantitative proteomic approach.”
LI-COR company has also introduced a new imaging software – Image Studio, can be used in conjunction with its Odyssey system. Users simply select a detection channel, and then press a button to get a picture. A new version of this software provides mm interval analysis, rather than cm intervals, while able to perform fully automatic and manual analysis.
Western blot re-create
In addition to the traditional Western blot techniques to fine-tune, ProteinSimple’s Simple Western system re-development was carried out by capillary electrophoresis so that the whole process is fully automated. In 2011, the company launched a Simon, plan to use the instrument fully replaced by Western blotting. Simon can be separated based on size, immunoassay, washing, chemiluminescent detection and quantification. Scientists only need to add the sample, and then leave, come back later you can get good data fully analyzed. Simon even beeps to inform the user the results came out.
Then in early 2012 the company launched the Sally, the instrument allows the user to carry out an experiment 96 Simple Western. The latest device is the September 2012 launch of Peggy, an analysis of 96 samples can be, and in accordance with size or charge separation proteins. Simple Western overcome the traditional western blotting Defects in manually, for example, or the lack of reproducible quantitative results. Further, in the conventional western blotting, multiplet detection has become a challenge. Researchers now to observe a sample by fluorescence in the two or three proteins, the transfer device 8 can be run at one time or four mini-gel medium sized plastic, but this is the traditional Western blotting can be achieved limit. Simple Western can multiplex analysis of protein in each capillary, thereby virtually eliminating the limitations of traditional Western blotting.
For oncology at Stanford University School of Medicine for teachers Alice Fan, Simple Western technology the most exciting thing is that it analyzes the ability of micro-level samples.
Fan collect tissue samples from patients for longer than 10 years, she has been waiting for that to make her tumor cell proteome analysis tools, but also need to take up the entire sample. “I’m looking for for years, want to have a device able to use a very small amount of tissue for Western blotting.” She explained.
In addition to help her save the previously saved outside the organization, Sally also allows Fan can use fine-needle aspiration method repeatedly extracted from a sample of the patient’s body, rather than do a comprehensive Biopsy. Fan hope that it will be able to help her patient’s tumor real-time observation of drug response. “In describing the different types of tumors, which is really beneficial for my research, which is great progress.”
Merck (Merck & Co) vaccine biochemistry team leader Richard Rustandi acquired at the end of 2011 with a Simon instruments. At the time, he did not have high expectations. But, he said, he got a surprise. “This instrument is great, we bought a few months later.”
According Rustandi introduction, although Simon was not perfect, but it can truly quantified. He went on to say, manually Western blotting typically have 35% to 45% of the variance ratio, but he and his lab Simon mutation rate can be controlled at below 10%.
With the Western blot technique to continue to evolve as researchers ultimately do not like babysitting, spending a lot of time looking after their experiments. “Their time is very important.” Simple Western product manager Peter Fung believes that “the higher utilization of your time, the more you can make a good research.”