Biacore Analysis

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purify@proteinx.com


Biomolecular Interaction Analysis on Biacore^(R) 1000 You know what analysis is. So let us guess why you come to this page: A. You got a new protein, try to identify up-stream/down-stream interaction partners. Recovery capable SPR (surface plasmon resonance) enables you to purify them from cell lysates, so you can analyze them through mass spectrum and sequencing. B. You are a start-up company, have made many Abs. You try to convince your customer that your Ab is better than ones from other supplier, you need real-time kinetic data to support your claim. C. You got several compounds. To decide which is a promising lead, you need to run systematic kinetic analysis, and SPR analysis is the best choice. D. You are studying a complex system, many components are involved. To understand the mechanism, SPR analysis is the only choice. E. You try to publish your paper, and thought that if you could include real-time interaction data in your paper, reviewers will be greatly convinced. F. You want to run biacore analysis at extreem conditions, for example, to get rid of mass transportation problem, you need high flow rate, one of our units is modified to run over 200ul/min (not 100ul/min limitation). G. You may... offers Biomolecular Interaction Analysis service by using Biacore^(R) 1000 (Five units are available). Our scientists are among the first generation of users back to early 90's (Xie Z, Reed JC. Methods Enzymol. 2000;322:266-74). We will assist you to fulfill your task. For individuals, our services range from confirming the interaction ("Yes or No" ) to performing detailed kinetic analysis (ka, kd, KD). Results will be presented in publishable form. This service is offered mainly to scientists who do not have time to go through this labor intensive analysis. There is a setting-up fee ($2800.00) for each project to cover equipment maintenance and initial assay development. The charge for each pair of interaction is $1200.00. The turnaround time is about 2 weeks. You need to provide 100~200ug of protein (95% pure). If you prefer, you may rent our equipment time at $28/hr (units are controlled by IBM Thinkpad, thus, you can take your dada with you, analyze them, and prepare the second day's run). For group (universities, companies, institutes), we offer site usage (you may renew it every year). Scientists without experience on Biacore analysis will be trained in our lab. Through this, if you feel this technology is good for your department, you may have confidence to set up your own facility since you already have trained scientists (good biacore analysis is dependent on both equipment (30%) and experienced scientists (70%). Biacore^(R) is a registered trademark of Biacore AB, ProteinX Lab is not affiliated with Biacore International, AB or any of its subsidiaries Purification of IbeA receptor that contributes to E. coli invasion of Brain One of puzzles in the development of E. coli meningitis is how it overcomes the blood-brain barrier. Recent experiments has shown that IbeA in cerebrospinal fluid isolates of E. coli is necessary for invasion of the brain microvascular endothelial cells (BMEC) that constitute the blood-brain barrier. 55Kd receptor of IbeA is identified in Bovine BMEC. However, the human receptor of IbeA is still unclear. (NV. Prasadarao et al. Identification and Characterization of a Novel Ibe10 Binding protein That Contributes to Escherichia coli Invasion of Brain Microvascular Endothelial Cells, Infection and Immunity, Mar. 1999, p. 1131-1138). We collaborate with researchers in USC to purify human receptors of IbeA by using Biacore. Purified IbeA was immobilized on CM5 chip, BMEC lysates were tested. Binding condition was optimized, then was used in affinity chromatography. Now, we have been able to identify a potential receptor of IbeA in human BMEC. Characterization of this receptor protein is underway. 1. IbeA used to purify receptor proteins 2. Complex from native IbeA affinity chromatography 3. Complex from denatured IbeA affinity chromatography Updates on 3/1/05 Purified receptor band has been sequenced, and the recombinant protein has been produced. It has been found that it interacts with apoptosis proteins, and induce the apoptosis of BMEC. This is a good explanation for E.coli invasion of brain. Pegylation and Biacore Analysis Pegylation has been used in pharmaceutical industry for enhancing the stability of proteins/peptides. Examining the effect of Pegylation on the binding kinetics of original activator/inhibitors is of interest. Biacore analysis measures real-time biomolecular interaction, which principle is based on the refractive index of solution near the surface. PEG has a very low refractive index (1.3 ), compared with BSA (1.9), thus there are few biacore analysis reports of pegylated peptides (small mass). We have run a successful analysis of pegylated peptides on Biacore 1000. The molecular weight of original peptide is 5Kd, and pegylated peptide is around 45Kd. Biacore Analysis and GST fusion protein purification You come to the fun part of Biacore analysis.