Introduction to Experimental Biophysics Biological Methods for Physical Scientists, Second Edition FOUNDATIONS OF BIOCHEMISTRY AND BIOPHYSICS SERIES Introduction to Experimental Biophysics: Biological Methods for Physical Scientists, Second Edition Jay L Nadeau Introduction to Single Molecule Biophysics Yuri L Lyubchenko Biomolecular Thermodynamics: From Theory to Application Douglas Barrick Biomolecular Kinetics: A Step-by-Step Guide Clive R Bagshaw An Introduction to Biophysics: Quantitative Understanding of Biosystems, Second Edition Thomas M Nordlund and Peter M Hoffmann Introduction to Experimental Biophysics Biological Methods for Physical Scientists, Second Edition Jay L Nadeau CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2018 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S Government works Printed on acid-free paper International Standard Book 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: CRC Press, Taylor & Francis Group, [2017] | Series: Foundations of biochemistry and biophysics Identifiers: LCCN 2017010261| ISBN 9781138088153 (hardback) | ISBN 1138088153 (hardback) | ISBN 9781498799591 (pbk ; alk paper) | ISBN 1498799590 (pbk ; alk paper) Subjects: LCSH: Biophysics Experiments Technique Classification: LCC QH505 N247 2017 | DDC 572 dc23 LC record available at https://lccn.loc.gov/2017010261 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com v Contents Series Preface xvii Preface xix Acknowledgments xxi Author xxiii Contributors xxv Chapter Introduction and Background Chapter Basic Molecular Cloning of DNA and RNA 43 Chapter Expression of Genes in Bacteria, Yeast, and Cultured Mammalian Cells 75 Chapter Advanced Topics in Molecular Biology 129 Chapter Protein Expression Methods 157 Joshua A Maurer Chapter Protein Crystallization Oliver M Baettig and Albert M Berghuis 187 vi Contents Chapter Introduction to Biological Light Microscopy 225 Coauthored with Michael W Davidson Chapter Advanced Light Microscopy Techniques 279 Coauthored with Lina Carlini Chapter Advanced Topics in Microscopy II: Holographic Microscopy 305 Coauthored with Manuel Bedrossian Chapter 10 Quantitative Cell Culture Techniques 325 Chapter 11 Semiconductor Nanoparticles (Quantum Dots) 361 Chapter 12 Gold Nanoparticles 395 Edward S Allgeyer, Gary Craig, Sanjeev Kumar Kandpal, Jeremy Grant, and Michael D Mason Chapter 13 Advanced Topics in Gold Nanoparticles: Biomedical Applications 429 Chapter 14 Surface Functionalization Techniques 453 Chapter 15 Electrophysiology 497 Coauthored with Christian A Lindensmith and Thomas Knöpfel Chapter 16 Spectroscopy Tools and Techniques 553 Chapter 17 Introduction to Nanofabrication 623 Orad Reshef Contents Glossary 643 Appendix A: Common Solutions 683 Appendix B: Common Media 689 Appendix C: Restriction Endonucleases 693 Appendix D: Common Enzymes 721 Appendix E: Fluorescent Dyes and Quenchers 723 Appendix F: Fluorescent Proteins 729 Index 731 vii http://taylorandfrancis.com ix Detailed Contents Series Preface xvii Preface xix Acknowledgments xxi Author xxiii Contributors xxv Chapter Introduction and Background 1 1.1 BASIC BIOCHEMISTRY 1 Molecules important to molecular biophysics Making use of functional groups 1.2 ENERGIES AND POTENTIALS 8 Biologically relevant energy scales Ionic bonds Ion–dipole interactions 10 Dipole–dipole interactions 11 Hydrogen bonds 12 The (strept)avidin/biotin interaction 14 1.6 TRANSLATION AND THE GENETIC CODE 26 1.7 PROTEIN FOLDING AND TRAFFICKING 28 1.8 ALTERNATIVE GENETICS 33 1.9 WHAT IS CLONING? 34 1.10 DESIGN OF A MOLECULAR BIOLOGY EXPERIMENT AND HOW TO USE THIS BOOK 35 BACKGROUND READING 40 Chapter Basic Molecular Cloning of DNA and RNA 43 2.1 INTRODUCTION 43 2.2 OBTAINING AND STORING PLASMIDS 45 1.4 CELLS 19 2.3 SELECTION OF AN APPROPRIATE E COLI AMPLIFICATION STRAIN; TRANSFORMATION OF E COLI WITH PLASMID 47 Transformation 47 Selection 48 Transformation efficiency 49 1.5 DNA, RNA, REPLICATION, AND TRANSCRIPTION 21 The structure and function of DNA and RNA 21 Replication 23 Transcription 25 2.4 PLASMID AMPLIFICATION AND PURIFICATION 49 Amplification 49 Purification 49 Measuring concentration and purity of extracted DNA 51 1.3 PRINCIPLES OF SPECTROSCOPY 17 What can be measured 17 How transitions are measured 18 750 Index obtaining and storing, 45–46, 46f Multiple cloning region (MCS), 159 purification, 49–51 Multiple cloning site (MCS), 56 RNA methods, 63–65 Multispectral techniques, 279–283 sequencing, 62–63 Mutagenesis site-directed mutagenesis, 66–68 multiple, 133–134 Southern blot (DNA)/ Northern blot (RNA), 65–66 techniques, site-directed, 66–68 Molecular Devices, 513, 622 Mutation, 25 Molecular mechanics, defined, 33 Molecular orbital calculations, 10 Molecular orbital theory, 9–10 Molecular weight cutoffs (MWCO), 176 Molecules, importance of, 1–7; see also Biochemistry Moloney murine leukemia virus (MMLV), 120 Monochromator, 413 Monodispersity, 195–198 Monolayers, techniques for characterizing surface, 462–470 contact angle, 464–465 ellipsometry, 463–464 interaction with reactive dyes, 463 other methods, 470 scanning probe microscopy, 466–469 X-ray photoelectron spectroscopy, 465–466 Monolayers using functional silanes or thiols, preparing, 454–457 Monomeric streptavidin, 17 Monomers of living systems, 4f Monosaccharides, 4, 4f Monovalent streptavidin, 17 Mother liquor, 190 Motor proteins, 84 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 335 N NADH, 288–289 N-(aminooxyacetyl)-N’-(D-biotinoyl) hydrazine (ARP), 475 Nanobodies, 295 Nanofabrication, 623–641 material deposition, 631–633 ALD, 633 CVD, 633 spin coating, 631–632, 632f sputter deposition, 632 TE and EBE, 633 metrology, 634–636 AFM, 635, 635f, 636f profilometry, 635 SEM, 634–635 overview, 623–624 patterning, 624–626 FIB, 626 lithography, 625–626 nanoimprint lithography, 626 resist, 624–625, 624f pattern transfer, 626–631 dry etching, 627–629, 628f Multicolor labeling and avoidance of autofluorescence, 380 lift-off process, 629–630, 629f, 630f Multielectrode arrays (MEAs), 540 template stripping, 630–631, 630f Multifunctional nanoparticles, examples of, 590, 590t wet etching, 626–627, 627f Multiphoton microscopy, 286 planar process, 623, 624f Index sample clean, keeping, 638–641 AMI wash, RCA clean, piranha etch, 639 amount of time, 639 cleanroom staff, nice to, 639–640 dedicated labware, 638 descumming, 639 before experimenting with own process, recreate old work, 640 minimum feature sizes, 638 N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (NHS/EDC), 411 Nickases, 150 Nicotinamide adenine dinucleotide, 258 Nicotine, 16 Nitrocellulose, 169 Nitroveratryloxycarbonyl (NVOC)–biotin, 482 NMR, see Nuclear magnetic resonance (NMR) Noise “nothing goes in bottle” rule, 639 reduction, 309 spinning resist on small samples, 640 sources of, 309–311 tolerances, 638 subtraction, 512 toolbox properly outfitted, 640 thin-film characterization, 636–637 Nomarski prism, 241, 242, 243 Nonagglomerating, 409 ellipsometry, 636 Noncoding DNA sequences, 63, 64f four-point probe, 637 Noncoding regions, 25 Raman spectroscopy, 637 Noncoding RNA, 63 XPS, 636–637 Nonhomologous end joining (NHEJ), 147, 149 XRD, 637 Nonionic detergents, 171 Nanografting, 482 Nanoimprint lithography, 626 Nanoparticle-facilitated local heating, 423 Nanotechnology Characterization Laboratory (NCL), 449 National High Magnetic Field Laboratory, 601 National Institute of Standards and Technology (NIST), 449 Native protein purification, 170 Natural protein arrays, 137 N-dodecyl-β-D-maltoside, 483–484 Near-field scanning optical microscopy (NSOM), 469 Nonlinear absorbance behavior, in fluorescent protein, 555, 556, 556f Nonnative protein purification, 170 Nonquaternary suppression, 587 Nonradiative recombination, 244 Nonsense mutation, 66 Nonspecific binding of biomolecules, preventing, 483–484 Northern blot (RNA), 65–66 NSOM, 482 N-tert-butyl-α-phenylnitrone (PBN), 598, 598f Neomycin, 100 Nuclear Bohr magneton, 595 Nerve growth factor receptor (NGFR), 354f Nuclear g-factor, defined, 582 Neuronal cultures, 107 Nuclear magnetic resonance (NMR), 582–592 Neuronal recording and stimulation, techniques for, 546 Neurotransmitter acetylcholine, 16 Neutravidin, 481 Newport, 621 751 examining QD surfaces with liquid-phase, example, 584, 585f overview, 582–584, 583f, 584f paramagnetic nanoparticles as MR contrast agents, 589–592 752 Index examples of multifunctional nanoparticles, 590, 590t Organic molecules, classes of, 1, 2f fluorescent paramagnetic iron oxide nanoparticles, 591 Organometallic synthesis, 365–366; see also Quantum dots (QDs) iron nanoparticles in agarose slab, 590, 592f MR images of quail embryos, 590, 592f solubilization with fluorescently labeled lipid micelles, 591, 591f pulse techniques and MRI, 587–589, 589f solid-state, 586–587, 587f Origami E coli strains, 163 Origin of replication (ORI), 43, 159 Osmium tetroxide, 382 Osmometer, 507 Ostwald ripening, 365, 405 Overlap integral, 567 in structural biology, 593 Nuclear Overhauser effect spectroscopy (NOESY), 581, 593 P Nuclease-deficient Cas9 (dCas9), 147 Packaging cell line, 115 Nutrient broth, 690 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 517, 519 Nyquist criterion, 295 O Ocean Optics, 622 Off-axis holography, 320 Off-target activity, 149 Oleic acid, 4f, 366 Oligonucleotides, Oncogenes, 120 Open complex, 25 O-phthaldialdehyde (OPA), 387 Optical path difference (OPD), 238 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), 519 PANTHER (Protein Analysis through Evolutionary Relationships), 141, 145f Paraformaldehyde, 345 Paramagnetic nanoparticles, as MR contrast agents, 589–592 examples of multifunctional nanoparticles, 590, 590t fluorescent paramagnetic iron oxide nanoparticles, 591 iron nanoparticles in agarose slab, 590, 592f MR images of quail embryos, 590, 592f solubilization with fluorescently labeled lipid micelles, 591, 591f Optical recording, 541–544 Particle sizer, 195 Optical theorem, 396 Partitions, 136 Optical transfection, 110 Passaging, 76 Optical waveguide lightmode spectroscopy and quartz crystal microbalance, 339–341 Patch-clamp amplifiers, 514 Ordinary epifluorescence microscopy, 287f Ore formers, 362 Organelle probes, 266–267; see also Fluorophores for cell labeling Organelles, 19 Organic dyes, traditional, 258–261, 259t–260t; see also Fluorophores for cell labeling Patch clamping, 502–504 Patch pipettes, 527–528 making, 528–529 Pathogenic retrovirus, 34 Patterning, 624–626 FIB, 626 lithography, 625–626 Index nanoimprint lithography, 626 Photobiotin, 481 resist, 624–625, 624f Photobleaching, 246, 247f Pattern transfer, nanofabrication, 626–631 Photoconvertable FP, 295 dry etching, 627–629, 628f Photodynamic therapy (PDT), 576, 577 lift-off process, 629–630, 629f, 630f Photolithography, 625 technique, 623 Photoluminescence, 362 template stripping, 630–631, 630f decay curves, 288 wet etching, 626–627, 627f QD, 370 PEG, 199, 199f, 200 Photoluminescent, 243 PEGylated Au nanoparticles, 450 Photomasks, 482 Perforated patch recordings, 503–504; see also Electrophysiology Photometric detectors, 255 Performance metrics, 319 Periplasm, 20 PerkinElmer, 621 Perrin equation, 565 Phage display, 131 Phage vectors, 129–132; see also Cloning techniques for large cloning Phalloidin, 264 Pharmacokinetic/pharmacodynamic (PK/PD) studies, 449 Phase contrast, 238–240, 240f Phase contrast microscopy, 326 Phenanthridine derivative, 260 Phosphate-buffered saline (PBS), 415 Phosphates, Phosphodiester, Phospholipids, 483 Phosphorescence, 244 Phosphotungstic acid (PTA), 591, 591f Photoactivatable FP, 295 Photoactivatable proteins, 581 Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), 294–296 Photomultipliers, 256 Photomultiplier tube (PMT), 255 Photon correlation spectroscopy, 195 Photon counting mode, 559 Photooxidation, 289, 291 of endosomes, 290 Photoswitchable FP, 295 Photoswitching mechanisms of FPs, 295–296 of synthetic dyes, 296 Photothermal therapy, 431 improving GNRT by addition of, 439 use of gold in, 432 Phycoerythrin (PE), 345 Physics of fluorescent molecules, 243–247 Physics of microscopy, 225–229; see also Biological light microscopy Pichia pastoris, 164 PicoQuant, 621 Piezoelectric transducer, 280 Pinhole aperture, 252 Piranha clean, 639 Piranha etch, 454 principle, 294 Piranha solution, 454 probe requirements, 295–296 Pi stacking, 16 753 754 Index Planar process, nanofabrication technique, 623, 624f Polyethylene glycol (PEG), 370, 409, 483 Plaques, 129 Polyethylene oxide (PEO), 483 Plasma cleaning, 639 Polylinker, 56 Plasmid restriction mapping Polymerases, 24 restriction enzymes, 52–53, 52t Polymers of living systems, 4f restriction fragments for ligation, separation of, 54–55, 56f Polymethyl methacrylate (PMMA), 625 screening purified DNA, 53–54 Plasmids; see also Molecular cloning of DNA/RNA Polynucleotides, Polypyrrole, 546 Polystyrene, 516–517 about, 44 Polysulfone, 516–517 amplification, 49 Polyvinylidene fluoride (PVDF), 169 for cloning and expression, 46f Posttranslational processing, 30 concentration calculation, 51–52 Potassium aspartate, 506–507 lyophilized, 46 Potassium gluconate, 506–507 obtaining and storing, 45–46, 46f Potassium methanesulfonate, 506–507 purification, 49–51 Precipitant, 190, 199 purity of extracted DNA, 51–52 Precrystallization test, 202 Plasmon resonance frequency, 402 Prescreening test, 202 Plasmons, 398 Primary amine, Plate count, 326 Primary cells, 21 Plating efficiency, 332 Primary culture, 90–97 Pluripotent stem cells, 95 Primary miRNA, 138 Point mutation, 66, 67f Primary structure of protein, 28, 30f Point scanning, 252 Primer dimer, 62 Point spread function (PSF), 294 Primers, mutagenic, 67 deconvolution techniques, 294 Princeton Instruments, 622 Polarization and DIC, 240–243 Prisms, 241 Polarized light microscopy, 240 Probe hybridization, 65 Polarizers, 559 Probes, spin, 596–598 Polyacrylamide gel, 167, 169 PBN, 598, 598f Polyacrylamide gel electrophoresis (PAGE), 194 spin labeling, 596, 597f Polybrene, 353 TMP and TEMPO, 597–598, 597f, 598f, 600, 600f Polychromatic imaging, 318 Probe sonicator, 171 Polydextran gel, 174 Profilometry, 635 Polydimethylsiloxane (PDMS), 309, 482 Prokarya, 19, 20f Index Promoters bacterial strains, 163 clearance, 25 insect cells, 164 for protein expression, 159–161, 160t mammalian cell, 164 Propidium iodide, 260 yeast, 164 Prospects for high-speed imaging, 413–414 expression system, 157–158 Prostate cancer, 438 expression vector selection Protease cleavage sites, 162 expression vector, 162–163 Proteases, 30 promoters, 159–161, 160t Protein protein tags, 161–162 arrays, 137 chromatography, 172 folding, 28–33 improving, 211–212 long polypeptide chain, microarrays, 137 overexpression in bacteria, 164 purity, 194–195 quantity, 198 size analyzer, 195 structure, 28, 30, 30f tags, 161–162 subcloning into expression vector, 163 fluorescent protein Dronpa, expression/purification of, 177–180 protein concentration, 176 protein expression, 164–166 protein isolation/purification native vs nonnative purification, 170–171 protein lysate, preparation of, 171–172 SDS-PAGE for checking protein expression about, 166–167 protein separation, 167–168 protein visualization, 168–170 Protein isolation/purification trafficking (in E coli), 32f, 33 native vs nonnative purification, 170–171 variability, 198–199 protein lysate, preparation of, 171–172 Protein concentration, 176 on crystallization, 202 Proteome, 137 Protochlorophyllide (Pchlide) oxidoreductase, 581 Protein Data Bank, 188 Proton donor, 14 Protein database (PDB), 189 Protoplasts, 79, 82 Protein expression methods Protospacer adjacent motif (PAM), 149, 149t buffer exchange, 175–176 Provirus, 114 chromatography “Pseudotransduction,” 353 about systems, 172–173 Pufferfish, 509 affinity chromatography, 173–174 Pulsed-field gel electrophoresis, 133 size exclusion chromatography, 174–175 Pulsed lasers, 285 DNA source, identification of, 158–159 Pulse techniques, MRI and, 587–589, 589f expression strain/cell line, selection of Pure phase objects, 309 755 756 Index Purification of fragments, 57 Quantitative real-time PCR (qPCR), 135–136 Quantum dots (QDs), 283, 287–292, 361–375 of membrane proteins, 171 and bleaching, 283 plasmid, 49–51 CdSe QDs tags, 161 Purified protein arrays, 137 Purines, 4, 7f CdSe/ZnS nanoparticle bioconjugate, characterization, 606–610; see also CdSe/ZnS nanoparticle bioconjugate Pyranine, 269 measurement of ROS from QDs using EPR, 599–600, 599f, 600f Pyrimidines, 4, 7f MPA-coated, 584, 585f solid-state NMR, 586–587, 587f thiol-capped, 584, 585f Q commercial, 373–375 QDs, see Quantum dots (QDs) Quail embryos, MR images of, 590, 592f conjugation to dopamine and quantifying the effects on fluorescence per molecule bound, 387–389 Quantifying bacterial growth and death, 325–330 determination of size and concentration, 367–369 bacterial growth curves, 325–328 EPR, measurement of ROS from, 599–600, 599f, 600f bacterial inhibition curves and modeling, 328–329 examining QD surfaces with liquid-phase NMR, example, 584, 585f IC50 and minimum inhibitory concentration, 329–330 quantifying bacterial concentrations, 325–326 Quantifying mammalian cells, 331–337 example experiment, 289 fluorescence emission from, 571 counting mammalian cells, 331–333 FTIR spectrum, under cap exchange from MSA to pyridine, 580, 580f end-point methods for mammalian cells, 333–336 orange-emitting, 567, 568f Quantifying viruses, 349–355 physics of, 361–365 titering adenovirus by optical density, 353 solubilization and biofunctionalization of, 370–373 titering adenovirus by plaque assay, 350–352 synthesis of, 365–366 titering lentiviral vectors by flow cytometry, 353–354 three-photon process and, 286 titering lentivirus using p24, 355 Quantum dots (QDs)–antibody conjugates, 377–378 titering phage by plaque assay, 350 Quantum dots (QDs) applications, 375–386 titering retroviruses expressing a selectable marker, 354–355 correlated fluorescence and electron microscopy, 381–383 titering viral vectors, 349–350 multicolor labeling and avoidance of autofluorescence, 380 Quantitative cell culture techniques QD delivery to living cells, 376, 378–380 example experiment, 345–347 QDs as biosensors, 383–386 flow cytometry, 341–343, 344f single-particle tracking, 375–376 quantifying bacterial growth and death, 325–330 Quantum dots (QDs)–streptavidin, 373, 374 quantifying mammalian cells, 331–337 Quantum mechanics, 8, quantifying viruses, 349–355 Quantum yields, 245 Index Quartz anisotropic crystal, 280 Reading frames, 28, 28f Quartz crystal microbalance and optical waveguide lightmode spectroscopy, 339–341 Real-time polymerase chain reaction (rt-PCR), 351 Quasi-elastic light scattering, 195 Quaternary structure of protein, 30, 30f Quenchers, fluorescent, 723t–727t Quenching, 247 Quenching, fluorescence, 561–562 biomolecular quenching constant, 562 collisional/dynamic, 561, 561f, 562, 572 examples of quenchers and their target fluorophores, 562, 563t static, 561, 561f, 562, 572 757 Recombinant DNA, 34 Reducing and oxidizing system (ROXS), 296 Reflected light, 233 Refractive index, 241, 309, 310 Relaxation spin–lattice (longitudinal), 587–588, 589f spin–spin (transverse), 587–588, 589f Relaxivity, defined, 589 Renilla luciferase, 385 Replication, 23–25, 24f; see also Biochemistry Reporter gene, 100, 101 Research grade microscope, 230 R Resists, 624–625, 624f, 631 Radiation therapy or radiotherapy (XRT), 432–439 in cells, 434–435 Resolution of optical system, 226; see also Biological light microscopy gold nanoparticle-assisted radiation therapy, see Gold nanoparticle-assisted radiation therapy (GNRT) Restriction endonucleases, 693–719 principles of, 432–433 Restriction fragments for ligation, separation of, 54–55, 56f Restriction enzymes, 52–53, 52t Radiosensitizers, 433 Restriction mapping, 52 Radio Society of Great Britain, 601 Restriction sites, 53 Raman spectroscopy, 637 Reticuloendothelial system (RES), 430 Raman tag addition, 420 Retroviruses, 34, 114, 114f; see also Quantifying viruses Rapid amplification of cDNA ends (RACE), 134 Reverse Northern blot, 66 Rapid protein expression, 166 Reverse transcriptase, 34 Ratiometric dye, 268 Reverse transcriptase PCR (rt-PCR), 134, 136 Ratiometric imaging, 268 Reverse transcription, 114 Rayleigh scattering, 195 Reversible photoswitching mechanism, 296 contaminations in spectrum, 560, 561f RH dyes, 541 Rayleigh’s criterion, 227 Rhodopsin, 581 RCA clean, 639 Ribosomes, 21 Reactive dyes, 262, 463 RMN, 620 Reactive oxygen species (ROS), 296 RNA measurement, from QDs using EPR, 599–600, 599f, 600f Reactive sputtering, defined, 632 double-stranded, 34 fingerprinting, 63 758 Index methods, 63–65 toolbox properly outfitted, 640 microinjection of, 105–108 Sample holders, for different geometries, 559, 559f single-stranded, 33, 64 Saturated fats, structure and functions of, 23f Scalar coupling, 583, 584 RNA-dependent DNA polymerase, 34 Scan head, 255, 256f RNA-induced silencing complex (RISC), 138 Scanning cysteine accessibility mutagenesis (SCAM) method, 67 RNA interference (RNAi), 107 mechanism, 138f RNA polymerase, 25 RNA-dependent, 34 RNMR, 620 Rocking curve analysis, 637 Rosetta and Rosetta 2, E coli strains, 163 Rosetta-gami strains of E coli, 164 Rotating turret mechanism, 279 Rotational transitions, 18, 18f Scanning electron microscopy (SEM), 382, 477, 634–635 Scanning near-field optical microscope (SNOM), 581 Scanning probe microscopy (SPM), 635, 635f Scanning transmission electron microscopy (STEM), 382, 383 Scanning tunneling microscopy (STM), 467, 468–469 on proteins, 470 Scattered radiation, 396 Schizosaccharomyces pombe, 79 ScienceSoft NMRanalyst, 620 SciFinder, 619 Secondary amine, S Secondary structure of protein, 28, 30f Saccharomyces cerevisiae, 81, 133 Secretory pathway, 32, 32f Salt, 200 Seeding, 211 Salt bridges, 518b Selectable marker, 43 Salting out, 200 Selection antibiotics in cloning, 43 Salt windows, solvent compatibilities of, 580, 580t Self-inactivating retroviral vector, 138 Sample clean, keeping, 638–641 Sephadex, 174–175 AMI wash, RCA clean, piranha etch, 639 Sequencing gel, 62, 63f amount of time, 639 Sequencing primer, 63 cleanroom staff, nice to, 639–640 Sequencing technology, 62–63 dedicated labware, 638 Series resistance compensation, 511–512 descumming, 639 Serum albumin, 335 before experimenting with own process, recreate old work, 640 Sharp electrode recording, 504 minimum feature sizes, 638 “nothing goes in bottle” rule, 639 spinning resist on small samples, 640 tolerances, 638 Short hairpin RNA (shRNA), 138 Shuttle vector, 76, 78f Signal-to-noise ratio (SNR), 309 Silane–biotin–streptavidin sandwich on SiO2 features on a Si chip, 477–483 Index assembling streptavidin, final characterization, and using the sensor, 480 biotinylation and blocking, 479–480 micropatterning, 482–483 observing and cleaning the substrate, 477–478 silanization, 479 variations on a theme, 481–482 Silanes or thiols, preparing monolayers using functional, 454–457 Silanization, 479 Silent mutation, 66 Silica shell, formation of a, 419–420 Silicon oil, 193 Silver staining, 169 Simulating annealing, defined, 593 SOC, media, 690–691 Sodium citrate, 409 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and protein purity, 194 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for checking protein expression; see also Protein expression methods about, 166–167 protein separation, 167–168 protein visualization, 168–170 SoftMax Pro imaging cytometer, 347 Software development kits (SDK), 318 Solid-state NMR, 586–587, 587f Single guide RNA (sgRNA), 147, 148f Solubilization, with fluorescently labeled lipid micelles, 591, 591f Single-mode optical fiber, 317 Solution quality, on crystallization, 203–204 Single-particle tracking, 375–376 Solutions, 683–688 Single-photon counting, TCSPC, 572, 574–575, 574f Sonotransfection, 112 Single-point ellipsometer, 636 Southern blot (DNA), 65–66 Single-strand binding proteins (SSB), 24 Space clamp, 505 Single-stranded RNA, 33 SPARC, 542 Single-walled carbon nanotubes (SWNTs), 546 Sparse matrix screens, 193, 204 Site-directed mutagenesis, 66–68, 67 Spatial light modulation (SLM), 309 Sitting drop experiment, 213 Spatial resolution, determination, 306 Sitting drop method, 190, 191t Speckle noise, 309 Size exclusion chromatography (SEC), 174–175, 406; see also Chromatography Spectral mask, 308 Skin stem cells, 96 “Slow” capacitance compensation, 511 Small interfering RNA (siRNA) data analysis, 141, 142t–144t, 145t drug resistance mechanism (example experiment), 139–141 principles, 138–139 secondary screening, 146 Small unilamellar vesicles, 172 Smoluchowski equation, 562 759 Spectral multiplexing, 419 Spectrofluorometer, 557–560, 558f, 559f Spectroscopic transitions, 18 Spectroscopy, principles of; see also Biochemistry about capability of measuring, 17–18 transitions, measurement of, 18–19 Spectroscopy tools and techniques, 553–611 CdSe/ZnS nanoparticle bioconjugate, characterization, 606–610 EPR, 610, 610f 760 Index FTIR, 607–609, 608f overview, 553 TCSPC, 609, 609f time-resolved absorption, 575–577 UV–Vis and fluorescence emission, 606–607, 607f EPR spectroscopy, 592–602 basic principles, 592–596, 594f, 595f, 596t instrumentation, 601–602, 601f, 601t, 602t measurement of ROS from QDs using, 599–600, 599f, 600f spin probes and traps, 596–598, 597f, 598f fluorescence spectroscopy, 557–570 anisotropy, 562, 563–567, 564f, 566f, 567f applications, 561–570 PDT, 576, 577 transient absorption measurements, 576, 576f ultrafast pump-probe transient absorption, 576, 577f time-resolved fluorescence emission, 570, 571–575 fluorescence lifetime decays, 571, 573f IRF, 572, 574f TCSPC, 572, 574–575, 574f UV–Vis absorbance spectroscopy, 554–557 caveats and sources of error, 560–561, 561f artifacts of drying, 556, 557f energy transfer, 567–570, 568f, 569t, 570f nonlinearities, fluorescent protein, 555, 556, 556f instrumentation, 557–560, 558f, 559f simplified light paths of, 555, 555f quenching, 561–562, 561f, 563t x-ray spectroscopy, 602–606, 603f, 603t–605t, 606f guiding principles, 553–554, 554f Spekwin, 620 IR spectroscopy, 577–582 Spermidine, 110 ATR spectroscopy, 580 Spherical aberration, 227 DRIFT spectroscopy, 580 Spheroplasts, 79, 82 energy bands of stretching and bending vibrations, 577, 578t Spin coating, 631–632, 632f FTIR spectrometer, Michaelson interferometer and, 579, 579f Spin–lattice (longitudinal) relaxation, 587–588, 589f FTIR spectrum of QDs under cap exchange from MSA to pyridine, 580, 580f Spin-orbit coupling, 593, 594 SpinCore Technologies, 622 Spinning sidebands (ssb), 586 spectrum for ethyl cyanoacetate, 577, 579f Spin polarization (spin density), 596 time-resolved, 581–582 Spin probes and traps, 596–598 transmission properties and solvent compatibilities, 580, 580t NMR, 582–592 examining QD surfaces with liquid-phase, example, 584, 585f overview, 582–584, 583f, 584f paramagnetic nanoparticles as MR contrast agents, 589–592, 590t labeling, 596, 597f PBN, 598, 598f TMP and TEMPO, 597–598, 597f, 598f, 600, 600f Spin relaxation, defined, 587 Spin–spin (transverse) relaxation, 587–588, 589f Splitting chemical shift, smaller than, 584, 584f pulse techniques and MRI, 587–589, 589f degenerate-energy electron spin states, 593, 594f solid-state, 586–587, 587f degenerate-energy nuclear spin states, 582, 583, 583f in structural biology, 593 hyperfine, 594, 595, 595f Index Spodoptera frugiperda, 164 Super optimal broth (SOB), 77t Sputter deposition, 632 Supersaturation, 189, 190 Static quenching, fluorescence, 561, 561f, 562, 572 Surface-enhanced Raman scattering, applications of gold nanoparticles in Stationary-phase culture, 164 Stem cells, 95 Stepper, 625 Steric hindrance, 12 Sterile alpha domain (SAM), 141 Sterile nanoparticles, 443 Sterile technique, 87 Stern–Volmer equation, 561–562 Sticky ends, 55, 55f Stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM), 294–296 principle, 294 probe requirements, 295–296 Stokes-Einstein equation, 196, 562 Stokes shift, 244 Streptavidin, 14, 17, 431, 483 Streptavidin–biotin, 485 Streptavidin-coated surface, 475 Streptococcus pyogenes, 150 Streptomyces, 14 Stretching vibrations, IR energy bands of, 577, 578t Stripping, 639 template, 630–631, 630f Strong confinement regime, 364 Structural biology, NMR in, 593 Subcloning, 59 into expression vector, 163 Sulfhydryl, Sulfo-N-hydroxysulfosuccinimide, 265 Sulforhodamine B (SRB), 433 Sulfosuccinimidyl ester, 374 SuperBlock solution, 480 761 introduction to Raman scattering, 416–419 protected Raman-active nanospheres, 419–420 SERS nanoparticles, 420–422 Surface functionalization techniques, 453–490 alkanethiol self-assembled monolayers, 457–461 functionalization of modified surfaces using cross-linkers, 470–475 controlling protein orientation, 473–475 types of cross-linkers, 470–473 preparing monolayers using functional silanes or thiols, 454–457 preventing nonspecific binding of biomolecules, 483–484 silane–biotin–streptavidin sandwich on SiO2 features on a Si chip, 477–483 assembling streptavidin, final characterization, and using the sensor, 480 biotinylation and blocking, 479–480 micropatterning, 482–483 observing and cleaning the substrate, 477–478 silanization, 479 variations on a theme, 481–482 some special considerations, 461–462 techniques for characterizing surface monolayers, 462–470 contact angle, 464–465 ellipsometry, 463–464 interaction with reactive dyes, 463 other methods, 470 scanning probe microscopy, 466–469 X-ray photoelectron spectroscopy, 465–466 testing the function of immobilized proteins, 484–491 electrochemistry, 485 762 Index enzymatic function, 485 Tetra-octylammonium bromide (TOAB), 404 ion channel function, 485 Tetrodotoxin (TTX), 509 specific binding: quantity and kinetics, 484–485 T7 expression system, 160 Surface plasmon resonance, 432 Theory of orbital formation, Surfactants, 366 Thermal cycler, 62 Surround or S-wave, 238 Thermal evaporation (TE), 633 Suspension cells, 89 Thermal scanning microscopy (TSM), 467 Synthetic dyes, 261, 296 ThermoFisher Scientific, 621 photoswitching mechanisms of, 296 Thin-film characterization, 636–637 ellipsometry, 636 T TA cloning, 61 Taguchi method, 628 T1 and T2 relaxation, 587–588, 589f TARQUIN, 620 TCSPC (time-correlated single-photon counting), 572, 574–575, 574f, 609, 609f four-point probe, 637 Raman spectroscopy, 637 XPS, 636–637 XRD, 637 Thioester, Thiol-capped QDs, liquid-phase NMR of, 584, 585f Thiols, 1, 296 Tellurium dioxide, 280 3D Interactive Chemical Shift Imaging (3DiCSI), 620 Telomeres, 133 Time-correlated single-photon counting (TCSPC), 290, 572, 574–575, 574f, 609, 609f Temperature, on crystallization, 203 Template stripping, 630–631, 630f TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy), 597–598, 597f, 598f, 600, 600f Time-gated imaging, 380 Time-resolved absorption, 575–577 PDT, 576, 577 Termination codons, 27 transient absorption measurements, 576, 576f Termination of transcription, 26 ultrafast pump-probe transient absorption, 576, 577f Terminator, 26 Terrific broth (TB), 77t, 691 Tertiary amine, Tertiary structure of protein, 28, 30f Tetracycline, 43 2,2,6,6-Tetramethylpiperidine (TMP), 597–598, 597f, 598f, 600, 600f 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), 597–598, 597f, 598f, 600, 600f Time-resolved fluorescence emission, 570, 571–575 fluorescence lifetime decays, 571, 573f IRF, 572, 574f TCSPC, 572, 574–575, 574f Time-resolved IR spectroscopy, 581–582 Titanium sapphire ultrafast systems, 576 TMP (2,2,6,6-tetramethylpiperidine), 597–598, 597f, 598f, 600, 600f Toluene, 556 Tetramethyl rhodamine (TMR), 258 TOPO cloning, 163 Tetramethylsilane, 583, 583f Total internal reflectance (TIRF), 287 Index Trafficking, protein, 31, 32f, 33 Trioctylphosphine (TOP), 587 Transcription, 25–26, 26f; see also Biochemistry Triphenylene molecular symmetry, fundamental anisotropy, 564, 564f bubble, 25 Transcriptome, 131 Trypan blue, 326 Transfection Trypsin, 89 defined, 75 Trypticase soy broth (TSB), 77t optimizing, 99 Tryptophan, 289 stable, 100–103, 269 Tube length, 226 Transfection of mammalian cells I about, 97–98 763 Turner bacterial strain, 163–164 T1-weighted and T2-weighted MRI, 588, 589f cationic liposomes, 98–100 electroporation of cell cultures, 105 example experiment, 103–105, 104 microinjection of DNA and RNA, 105–108 stable transfection, 100–103 Transferrin, 264 Transfer RNA (tRNA), 26 Transformation amplification strain of E coli, 47, 48f defined, 75 of E coli strains, 76 U Ultrafast pump-probe transient absorption, 576, 577f Ultrafast Systems LLC, 621 Ultrasonic acoustic wave, 280 Ultraviolet–visible (UV–Vis) absorbance spectroscopy, 51, 325 Ultraviolet–visible (UV–Vis) “absorbance” techniques, 398 Ultraviolet–visible (UV–Vis) spectrometer, 165 Unsaturated fats, Uranyl acetate, 382 UV-Visible absorbance spectroscopy, 18, 408, 554–557 efficiency, 49 artifacts of drying, 556, 557f Transient transfection techniques, 98 Translation in E coli, 26–28, 27f; see also Biochemistry CdSe/ZnS nanoparticle bioconjugate, characterization, 606–607, 607f Transmission diffraction grating, 280 nonlinearities, fluorescent protein, 555, 556, 556f Transmission electron microscopy (TEM), 367, 383, 482–483 simplified light paths of, 555, 555f Transmission properties, IR, 580, 580t Transmitted light, 233 Traps, spin, 596–598 PBN, 598, 598f spin labeling, 596, 597f TMP and TEMPO, 597–598, 597f, 598f, 600, 600f V Valinomycin, 521 Van der Waals interactions, 12 Vapor diffusion, 190–193, 191t; see also Macromolecules, crystallization of Trichloroacetic acid (TCA), 176 Varian, 621 Trichoplusia ni, 164 Vesicular stomatitis virus G glycoprotein (VSV-G), 355 Tricoctylphosphine oxide (TOPO), 366 Vibrational transitions, 18, 18f 764 Index Vibrations, on crystallization, 203 Xenopus oocytes, 285 Viral protein, 350 X-ray crystallography, 215; see also Crystallization of proteins Viral vectors, 108, 113 Visibility map of hologram, 320 Voltage-clamp mode, 504–505 Voltage-sensitive dyes (VSD), 269, 343, 541 Voltage-sensitive fluorescent proteins (VSFPs), 542 Voltage setting, 255 X-ray diffraction (XRD), 637 X-ray free-electron laser (XFEL), see Crystallization of proteins X-ray photoelectron spectroscopy (XPS), 636–637 X-ray spectroscopy, 602–606, 603f, 603t–605t, 606f XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)2Htetrazolium-5-carboxanilide), 335 W Warner Instruments and Harvard apparatus, 513–514 Y Water Raman test, 560 Yeast, expression strains for, 164 Western blotting, 169, 169f Yeast artificial chromosomes (YAC), 133; see also Cloning techniques for large cloning Wet etching, 626–627, 627f Wheat germ agglutinin (WGA), 415 Yeast cells expression of genes, 79–84 Whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), 294 Yeast expression systems, 158 Whole-cell recording Yellow fluorescent protein (YFP), 271 on cells transfected with K+ channels and GFP, 532–535; see also Electrophysiology Young double-slit experiment, 318 2× YT, nutrient medium, 691 step-by-step guide to performing a, 530–532 Widefield fluorescence microscopy, 280–281 Z Wollaston prism, 241 Wurtzite, 361 Zeeman effect, 593, 595 Zernicke phase contrast, 308 X Zinc blende, 361 Zinc finger nucleases, 147 Xanthene dyes, 267 .. .Introduction to Experimental Biophysics Biological Methods for Physical Scientists, Second Edition FOUNDATIONS OF BIOCHEMISTRY AND BIOPHYSICS SERIES Introduction to Experimental Biophysics: Biological. .. An Introduction to Biophysics: Quantitative Understanding of Biosystems, Second Edition Thomas M Nordlund and Peter M Hoffmann Introduction to Experimental Biophysics Biological Methods for Physical. .. Biophysics: Biological Methods for Physical Scientists, Second Edition Jay L Nadeau Introduction to Single Molecule Biophysics Yuri L Lyubchenko Biomolecular Thermodynamics: From Theory to Application