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The last exercise of the course consists of posting 4 questions, one for each of the genomics, proteomics, biointeractions, and bioimaging units. Add your questions in the form of "comments" to this blog thread.
Among the questions that are correctly formulated, several of them will be randomly chosen. The selected questions will be worth 25% of the final exam.
Due date, May 8th before class.
The other components of the final exam will include (i) recycled/modified questions taken from previous exams, presentations, exercises (50%), new questions (25%).
Komatsu, et al. J. Am. Chem. Soc., 127 (31), 10798 -10799, 2005.
http://pubs.acs.org.floyd.lib.umn.edu/cgi-bin/abstract.cgi/jacsat/2005/127/i31/abs/ja0528228.html
Scanning Electrochemical Microscopy of Model Neurons: Constant Distance Imaging
Kurulugama,R.T.;Wipf.D.O.;Takacs,S.A.; Pongmayteegul,S.;Garris,P.A.;Baur,J.E. Anal.Chem,2005,77,1111-1117
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" Dual-Mode Nanoparticle Probes for High-Performance Magnetic Resonance and Fluorescence Imaging of Neuroblastoma" Jae-Hyun Lee, Young-wook Jun, Soo-In Yeon, Jeon-Soo Shin, and Jinwoo Cheon Angew Chem Int Ed 2006, 45, 8160-8162.
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Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy
Thomas Hellerer, Claes Axang, Christian Brackmann, Per Hillertz, Marc Pilon, and Annika Enejder
www.pnas.org/cgi/doi/10.1073/pnas.0703594104
Download file for a complete version of the text below.
Comments on the Answers to Exercise, 20, 21
Chem 8157 – Spring 2008
Every group did manage to report a Kd value. The values are summarized below.
Question 8. The only issue of concern is the determination of the lowest and highest Kd that could be experimentally measured for a similar system (question 8).
1. Perhaps, the most satisfactory answer (without actually doing the experiment with the new system, would come from assuming that the same concentration of ligand are used. Then, determine the extreme variations that could be measured.
For example, assign the minimum separation in the CE peaks to the highest concentration of AT. Indeed, you then need to assume that other AT concentrations have even smaller undetectable peak separations. At the other extreme, you may consider that the lowest concentration causes a peak shift that it is barely within the migration time window of the electropherogram because the shift is very large. You would then need to assume that other peaks would be out of this range.
2. The other possibility is to base your estimates on the errors associated with Kd. Please note that the Kd comes from the slope in a Scatchard plot or from the error in the ‘e’ coefficient in the binding isotherm. Consider then, for the Scatchard plot for example, that one can detect a slope that is as small as 3x Std. Dev. above a slope of zero. The other extreme comes from considering the the slope is infinity. One then need to consider the smallest measurable value of the x-axis and the highest measurable value on the y-axis.
Similar arguments would hold when using the ‘e’ coefficient to estimate the limits.
Keep in mind that these are only estimates!
Why are the values so different?
Data analysis – the quality of the data limits their resolution; a large error comes from the actual measurements. This factor is particularly important for the Gel, AT experiments. It does not matter whether an x-reciprocal plot or a NLR plot is used.
The technique – CE determinations provide migration time data with better precision than the gel data.
Heparin heterogeneity – It appears that the CE and the Gel reports used different types of heparin.
7) Our Kd value of 7.62 nM was calculated and is in comparison to the Kd values obtained for the paper of 16nM and 12 nM (calculated for two different solvents). The %error of our value with the paper's values are 52.5% and 36.7%. Overall we felt are calculations are solid but with sources of error: we did not measure any of the mobilities present at 1nM [AT], we may have measured the points and normal band with a different method than Lee et. al., and our measurements are limited by the resolution of the photograph.
8) To determine the minimum Kd value that our method could reasonably detect, we used the standard error in the slope of our Scatchard Plot. We took the standard error (.009) multiplied it by the square root of the number of observations (8) to get the standard deviation (.0025). We took three times the standard deviation (.075) and subtracted it from our slope value (-.1313), which gave us -.2063. This represents the lowest possible slope that we can accurately measure. Since the slope=1/(-Kd), the lowest possible Kd we can measure using this method is 4.8 nM. We used a different method to determine the highest possible Kd obtainable. From the gel given in the paper we measured the furthest possible migration that we thought we could reasonably measure at a concentration of 1000 nM. From this measurement we determined the R factor for this Kd and concentration (.1319). For our point we assumed that at a concentration of 1 nM the R factor is going to be really low, since the amount of hepatin bound will be so small it will essentially be like free hepatin moving through the matrix, and will therefore have an R factor of close to zero. To get an accurate Scatchard Plot we needed to assume that our R factor would not be smaller than the R/[Free Protein] factor. This meant estimating an R factor of .00014, which is very close to zero. We created another Scatchard Plot for these two points and from the slope of that graph determined the maximum Kd, which we found to be approximately 16210 nM.
We were able to calculate from the affinity gel electrophoresis results, a dissociation constant of 1.3 nM for bFGF which compares fairly with the literature value of 2 nM. We calculated the dynamic range to be from 0.3 nM up to 35 nM.
Example of an exam on biointeractions.
New questions were added on April 26, 2008.
This is a compilation of general questions about the material that was covered in Unit 4.
Feel free to post more questions here. If you know the answer, free to answer any of the questions posted by others. Edgar will moderate the answers and provide additional comments when needed.
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Please see attached PDF.
http://www.micab.umn.edu/faculty/Bond.html
Capillary Electrophoresis-SELEX Selection of Aptamers with Affinity for HIV-1 Reverse Transcriptase
Renee K. Mosing, Shaun D. Mendonsa, and Michael T. Bowser
Analytical Chemistry, 2005, 77, 6107-6112
http://pubs.acs.org/cgi-bin/article.cgi/ancham/2005/77/i19/pdf/ac050836q.pdf
Studies on the interation of protein with acid chrome blue K by electrochemical mehod and its analytical application
Wei Sun, Junying Han, Kui Jiao, Lude Lu
Bioelecrochemistry 68(2006) 60
Yang, T. et. al., JACS. 2003
Title: ssDNA Aptamer-Based Surface Plasmon Resonance Biosensor for the Detection of Retinol Binding Protein 4 for the Early Diagnosis of Type 2 Diabetes
Author: Su Jin Lee,† Byung-Soo Youn,‡ Ji Woo Park,‡ Javed H. Niazi,† Yeon Seok Kim,† and Man Bock Gu*,†
Journal: Analytical Chemistry
Link to this paper: http://pubs.acs.org.floyd.lib.umn.edu/cgi-bin/asap.cgi/ancham/asap/pdf/ac800050a.pdf
Gold Nanohole Array Substrates as Immunobiosensors
John C. Sharpe, John S. Mitchell, Ling Lin, Nemanya Sedoglavich, and Richard J. Blaikie
Anal. Chem. 2008,80,2244-2249
Zhang et. al., Biochemistry, 2007
" A Fluorophore-Based Bio-Barcode Amplification Assay for Proteins "
Byung-Keun Oh, Jwa-Min Nam, Seung Woo Lee, and Chad A. Mirkin Small 2006, 2, 103-108
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Aptamer-Modified Monolithic Capillary Chromatography for Protein Separation and Detection
Qiang Zhao, Xing-Fang Li, and X. Chris Le
http://dx.doi.org/10.1021/ac702567x