Bio-Layer Interferometry (BLI) and Surface plasmon resonance (SPR) are two well-established techniques for detection and monitoring biomolecular interactions in real time. Used orthogonally, they can be powerful and complementary tools in basic research, drug discovery and development, and downstream bioprocessing.
While both SPR and BLI utilize light to make real-time characterizations of biomolecular interactions, BLI enables significantly higher throughput and flexibility by running up to 96 samples in a parallel.
The Octet BLI-based platform brings biosensors to the sample, rather than the other way around. Surface plasmon resonance systems rely on flow — which requires complicated microfluidics — to bring the sample to the sensor. No microfluidics means:
Read 8, 16, 48, or even 96 samples at a time. Complete a 32×32 binning experiment in less than 8 hours on the Octet HTX system, compared to 30+ hours on a 4-channel SPR system.
Samples are conserved with >99.9% available for recovery, so they can be re-assayed with different biosensors or transferred to another assay.
SPR measurements rely on the refractive indices of the solution and analyte. If there isn’t an adequate difference in the refractive indices, mass can’t be measured. And SPR is less tolerant of samples containing high levels of glycerol and DMSO. BLI measures only what’s bound to the biosensor which means it’s relatively indifferent to the refractive index of the solution. This lets you work directly in crude, unpurified samples with the Octet system.
Biacore vs. Octet®
|Detection technology||Surface Plasmon Resonance(SPR)||Bio-Layer Interferometry (BLI)|
|Signal read-out||Refractive index||Wavelength shift|
|Throughtput||Relatively high throughput||Up to 8 assays in parallel per 96-well plate allowing high throughput|
|Sample compability||Less tolerant of samples containing high levels of solvent||Insensitive to matrix pH or changes in refractive index and wide solvent tolerance|
|Sample recovery||Relatively high sample recovery||Full sample recovery|
|Sample analysis time||12 hours for 3 samples (SPR“B”)||24 minutes for 96 samples (Octet®RED384)|
|Cut costs per data point||Relatively high||Relatively cheap|
Source from: http://www.fortebio.com//octet-bli-spr.html
Intrigued by what Octet systems and BLI can do for you? Our biomolecular interaction analysis service based on BLI technology can meet your specific needs and deliver very accurate analysis of your samples. To get a price quotation, just fill out our Quotation Form and email to Sales@BiologicsCorp.com – we will get back to you in 24 hours.
Protein-protein interactions (PPIs) are the basis of many important cellular processes such as signal transduction, molecular transport and various metabolism pathways, while aberrant PPIs are the basis of multiple aggregation-related diseases, such as Alzheimer’s disease, and may lead to cancer. Therefore, PPIs have been studied extensively in the area of bioscience and medical research. Here, we review the major contemporary approaches utilized for detection and analysis of protein protein interaction.
|An immunoprecipitation experiment designed to affinity-purify a bait protein antigen together with its binding partner using a specific antibody against the bait.|
|Pull-down Assays||An affinity chromatography method that involves using a tagged or labeled bait to create a specific affinity matrix that will enable binding and purification of a prey protein from a lysate sample or other protein-containing mixture.|
|Yeast two-hybrid (Y2H)||Monitor complex formation through transcriptional activation of reporter genes.|
|Far-Western Blotting||Similar strategy to Western blotting with one key difference. The antibody probe in a typical Western blotting detection is substituted with an labeled bait protein as the probe.|
|Tandem affinity purification-mass
|TAP-MS is based on the double tagging of the protein of interest on its chromosomal locus, followed by a two-step purification process and mass spectroscopic analysis.|
|Protein microarrays||Microarray-based analysis allows the simultaneous analysis of thousands of parameters within a single experiment.|
|Bio-Layer Interferometry(BLI)||Change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time.|
|Surface Plasmon Resonance (SPR)||SPR angle changes with surface refractive indexes, which is in direct proportion to the molecular mass of the biomolecule attached to the metal surface.|
Co-immunoprecipitation (Co-IP) is a popular technique to identify and validate physiologically relevant protein-protein interactions. By using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein, Co-IP is applied to screening novel protein-protein interactions or confirming the existence of protein-protein interactions.
In Co-IP proteins interact in a non-denaturing condition which is almost physiological. However, low affinity or transient interaction between proteins may not be detected. On the other hand, the result of Co-IP could not determine whether the interaction is direct or indirect, since the possibility of involvement of additional proteins could not be ruled out.
Pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins. It can be used for confirmation of existing protein-protein interactions discovered by other techniques or initial screening to identify novel protein-protein interactions.
The basic principle of pull down assay is to utilize a tag fused protein (such as GST-tag, His-tag and biotin-tag) immobilized to affinity resin as the bait protein. Proteins binding to the bait protein (prey protein) can be captured and “pulled down” when the target protein or cell lysate flows through. By subsequent elution and analysis using Western Blot or Mass Spectrometry, a predicted interaction can be confirmed or previously unknown interactions can be discovered.
The two-hybrid system is one of the most widely used methods to screen or confirm protein–protein interactions. Two protein domains are required in the Y2H assay which will have two specific functions: (i) a DNA bindingdomain (DBD) that helps binding to DNA, and (ii) an activation domain (AD) responsible for activating transcription of DNA. Both domains are required for the transcription of areporter gene. Y2H analysis allows the direct recognition of PPI between protein pairs. However, the method may incur a large number of false positive interactions. On the other hand, many true interactions may not be traced using Y2H assay, leading to false negative results.
Far-western blotting is a molecular biological method which is based on the technique of western blotting to detect protein-protein interaction in vitro. While usual western blotting uses an antibody to detect a protein of interest, far-western blotting uses a non-antibody protein, which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is rather employed to detect protein protein interactions.
TAP tagging was developed to study PPIs under the intrinsic conditions of the cell. This method is based on the double tagging of the protein of interest on its chromosomal locus, followed by a two-step purification process. Proteins that remain associated with the target protein can then be examined and identified through SDS-PAGE followed by mass spectrometry analysis, thereby identifying the PPI collaborator of the original protein of interest.
Protein microarrays are rapidly becoming established as a powerful means to detect proteins, monitor their expression levels, and probe protein interactions and functions. A protein microarray is a piece of glass on which various molecules of protein have been affixed at separate locations in an ordered manner. Protein microarrays have seen tremendous progress and interest at the moment and have become one of the active areas emerging in biotechnology.The objective behind protein microarray development is to achieve efficient and sensitive high-throughput protein analysis, carrying out large numbers of determinations in parallel by automated process.
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