Frequently asked questions

How can I check sensitivity and performance of Bio-Logic systems?

Bio-Logic staff will be pleased to organize a demonstration session. It can be organized in the French offices, in one of our demonstration sites or in your laboratory depending on configuration.

250 μs dead time in Circular Dichroism mode:
Cooperative Sub-Millisecond Folding Kinetics of Apomyoglobin pH 4 Intermediate
Sebastien Weisbuch, Francine Gérard, Marielle Pasdeloup, Jéremy Cappadoro, Yves Dupont, and Marc Jamin
Biochemistry 2005, 44, 7013-7023
http://pubs.acs.org/cgi-bin/article.cgi/bichaw/2005/44/i18/pdf/bi047372v.pdf

550 μs dead time in fluorescence mode:
Rapid mixing methods for exploring the kinetics of protein folding
H.Roder, K.maki, H.Cheng, M Ramachandra Shastry Methods 34 (2004) 15-27

200 μs dead time in fluorescence mode:
THE MECHANISM OF THE REVERSE RECOVERY-STEP, PHOSPHATE RELEASE AND ACTIN ACTIVATION OF DICTYOSTELIUM MYOSIN II
Journal of BioLogical chemistry, 2008
http://www.jbc.org/cgi/reprint/M708863200v1.pdf

Is it easy to switch from absorbance to fluorescence/light scattering position using MOS-200?

The standard version MOS-200 includes one PM tube which is optimized to work both in the UV and visible region from 140 nm to 900 nm. The Photomultiplier tube can be used for absorbance, fluorescence, and also for far UV-Circular dichroism ! Changing from absorbance position to fluorescence position is done in less than 20 seconds, with a single control knob.

A second PM tube can be added as an additional detection channel. With an additional channel the user can measure 2 fluorescence, anisotropy or fluorescence/absorbance or CD/absorbance/fluorescence values simultaneously. It is not necessary to buy extra options to get these detection techniques activated.

No realignment of optics is necessary with MOS-200 so changing configuration is as simple as clicking a mouse!

How many type of observation cells are available? Also, can I use the same cell for absorbance and fluorescence measurements?

Bio-Logic proposes 8 different cells and most of them have two different pathlengths. For example TC-100-10 can be used in absorbance mode with 1 cm or 1 mm light path. The change of cuvette is also done in less than one minute.

Pathlength of 0.8 mm; 1.5 mm; 1mm; 2 mm; 1 cm are available.

Most of the cells have windows on 4 sides so it is possible to do alternatively (or simultaneously) absorbance and fluorescence without changing the cell.

Our system were designed to be modular and to offer unique experimental possibilities so user can change rapidly cell if necessary, you could also use capillaries for some special applications. We have two families of cuvettes. Our FC cell offers 0.8 mm, 1.5 mm or 2 mm light path in fluorescence and absorbance modes. The volume of the FC-15 cell is 30 μl, and the light path is 1.5 mm. There is absolutely no compromise on sensitivity. If absorbance is measured at the same time then the sensitivity of our detector will be more than sufficient to perform absorbance measurements. As a reference, noise level is absorbance mode is 5 10-5 AU at 1 ms integration time.

Our cells with 0.8 mm light path ( FC-08 and microcuvette) are the best cell on the market for inner-filtering effect and also for low dead time specifications.

The TC cuvettes ( 5 mm or 10 light path), have mean light paths of 3.5 mm or 5.5 mm light path when used in fluorescence mode.

Our policy is to provide the best cuvettes for the purpose and not to limit user to two different cells. More details at ‘http://www.bio-logic.info/rapid-kinetics/mixers.html#mixers_7

Is the light path of FC-08 cell really 0.8 mm?

YES! The illumination is done along a 0.8 mm x 0.8 mm axis so real light path in fluorescence (or absorbance mode) is 0.8 mm. In case you hear about other demonstration based on calculations introducing a 12.5 mm value, then just be aware that 12.5 mm is the half height of the cell and is thus not related to optical light path. Please feel free to contact us to get information on cell design so you can check by yourself.

Can I run time-resolved spectra with my MOS-200/M ? How fast is the acquisition?

The MOS-200/M has a motorized scanning monochromator and can do time-resolved absorbance spectra (or fluorescence excitation spectra). In the wavelength tracking mode the user can program a series of measurements at different wavelengths and then edit the 3 dimensional data (wavelength, absorbance, time) for Global fitting analysis. Our PM tube can measured one point every 10 μs, this is the fastest data acquisition time you can get between two spectra!

Another method is to use a Diode array detector. The advantage of diode array (compare to PM tube) is the sample economy as only one experiment is necessary to collect all data. The disadvantage (compare to PM tube) is sensitivity so the fastest acquisition time is 0.8 ms per spectrum. Our diode arrays are the fastest one on the market, 3 models can be proposed for all applications from UV to near IR.

Is it easy to remove bubbles during loading process?

Our standard 10 ml syringes offer the best dead time specifications in the industry. The priming lines are vertical (not ‘near horizontal ceiling’) so bubbles can be removed very easily during the filling process. The top of syringe is designed with a shape that allows the user to purge bubbles very easily. 10 ml is the maximum volume you can load in a single syringe, it does not mean it is necessary to put 10 ml inside. Syringes are controlled individually by independent drive mechanisms and may be loaded with the optimum volume for the individual syringes

What is the drive volume of Bio-Logic stopped-flow?

The driving volume is freely and independently controllable form software and it depends on cuvette size. Mininum injection volume using 10 ml syringes is 50 μl. For high mixing ratio a small syringe can be installed without compromising dead time, and you can then inject 10-15 μl with one syringe. The Bio-Logic SFM allows samples to be saved by optimizing mixing sequence. With multiple syringe volumes to choose from and Bio-Logic’s completely independent syringe control, you are not limited to factory set 1:5 or 1:10. You can freely select the optimum ratio for your experiment ( if 1:7.6 is what you need, then just do it !)

What is the dead volume of Bio-Logic stopped-flow?

Dead volume of SFM-20 is 50 μl. Dead volume of SFM-300/400 is 150 μl. If you need to refill driving syringes (with same solution) during one experiment then you do not need to replace this volume. If you need to change concentration between shots, you do not need to refill the syringe as you can just change mixing ratio from the software. There is NO risk of dilution. The Bio-Logic mixer design also lets you save samples: for example if you need to try 10 concentrations then total priming volume would still be 150 μl with SFM-300.

Can I use high mixing ratio ( > 1:10) with Bio-Logic stopped-flow?

The best answers are the many results published by our customer for more than 20 years. Scientists doing folding-unfolding experiments have selected and praised our instruments precisely because of their dilution performances. You can find many of the publications on our website. An example is selected below: in this work the users were using a 1:40 mixing ratio.

Interactions of non detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation
L.Vuillard, M.Goldberg
Eur.J.Biochem.256, 128-135 (1998)

Accuracy of mixing ratios is always demonstrated by our application specialists when doing system installations. It can be checked at any times using a dye dilution in your experimental viscosity conditions. If it works with a dye test then there is absolutely no reason why the mixing ratio can be wrong later on !

Each syringe has a separate and independently controlled drive motor. The drive ratio of the motors and controllers give step resolution in the nanoliter range. It is true that motors needs to accelerate to reach the desired flow rate, but the volume of one microstep in on the nanoliter time scale, and the volume pushed at each shot is in the 50 μl range. This means the acceleration is achieved in few microsteps continuous movement will be reached after few hundreds of nanoliters. So continuous flow is achieved from the first microliters and mixing ratio and flow rate are guaranteed to be accurate. The first microliters pushed into the cell are going to waste tube directly so there is no interference with customer data.

Only Bio-Logic offers a full flexibility on mixing ratios with no reason to lock mixing ratio on fixed values (for example 1:10 or 1:25)

How easy is it to run concentration dependence studies with SFM-300/400?

Mixing ratio can be changed by one click in the software, so a full series of concentration can be done in few minutes without refilling the system. This is a unique feature offered by Bio-Logic and this is possible thanks to our unique independent stepping motor technology.

A classical and old way to do concentration dependence studies is to do dilution manually, to empty and then to refill system at the end of each experiment. This procedure consumes time, sample and energy when compared to executing automatic dilution for a similar precision!

Is the mixing efficiency identical when changing mixing ratio?

Yes. The Ball mixer was specially designed to create turbulent flow with different mixing ratio so mixing efficiency will be similar whatever the ratio used. So SFM-300 and SFM-400 can be used from 1:1 to 1:100 with the same efficiency. Turbulent mixing is also guaranteed when using solvents, when mixing viscous solutions (up to 2000 cP) and when mixing solutions of different viscosities.

How can I change ageing time when doing double jump experiments?

Thanks to independent motors the user controls exactly the flow rates delivered by the motors and they can be changed by a single click in the software. The system can be operated in continuous or interrupted flow methods depending on experimental requirements. Delay lines with different volumes are also supplied so you can do quench-flow or high mixing ratio in double mixing experiments.

Our SFM-400 is also the only system in the world with 3 mixers, so triple mixing experiments can be done (concentration dependence studies and double jump can be done at the same time)

How can I fit my data?

Data generated by Bio-Logic stopped-flow can be directly analyzed in Biokine. Also, data are automatically saved as text files so they can be easily exported to external for your convenience.

What is the key benefit of SFM-400?

SFM-400 is the only system in the world having 4 syringes and 3 mixers. This is a unique system to do triple mixing experiment in stopped-flow mode and double mixing/quench flow experiments ( H/De exchange experiments for example).

I have read statistics comparing the number of publications referring to stopped-flow worldwide. It was indicated that only 10-12 papers were published by Bio-Logic instruments in 2005. Should I believe this number?

One of our application specialists spend 3 hours on internet searching papers referring to our stopped-flow in 2005. 47 papers were found during this time. Of course more can be found spending more time but it just give you good indications about the credibility of statistics:

1/ The reconstitution of unfolded myoglobin with hemin dicyanide is not accelerated by fly-casting. M. Crespin, B. Boys, L. Konermann FEBS Letters, Volume 579, Issue 1, 2005, Pages 271-274

2/ An On-pathway Hidden Intermediate and the Early Rate-limiting Transition State of Rd-Apocytochrome b562 Characterized by Protein Engineering Zheng Zhou, Yingzi Huang and Yawen Bai Journal of Molecular Biology Volume 352, Issue 4, 30 September 2005, Pages 757-764

3/ Kinetic Partitioning During Folding of the p53 DNA Binding Domain James S. Butler and Stewart N. Loh Journal of Molecular Biology Volume 350, Issue 5, 29 July 2005, Pages 906-918

4/ The Average Conformation at Micromolar [Ca2+] of Ca2+-ATPase with Bound Nucleotide Differs from That Adopted with the Transition State Analog ADP•AlFx or with AMPPCP under Crystallization Conditions at Millimolar [Ca2+] Martin Picard, Chikashi Toyoshima, and Philippe Champeil J. Biol. Chem., Vol. 280, Issue 19, 18745-18754, May 13, 2005

5/ Amyloid Formation of a Protein in the Absence of Initial Unfolding and Destabilization of the Native State Gemma Soldi, Francesco Bemporad, Silvia Torrassa, Annalisa Relini, Matteo Ramazzotti, Niccolò Taddei and Fabrizio Chiti Biophysical Journal 89:4234-4244 (2005)

6/ Amyloid Formation from HypF-N under Conditions in which the Protein is Initially in its Native State Giordana Marcon, Georgia Plakoutsi, Claudio Canale, Annalisa Relini, Niccolò Taddei, c, Christopher M. Dobson, Giampietro Ramponi and Fabrizio Chiti Journal of Molecular Biology Volume 347, Issue 2, 25 March 2005, Pages 323-335

7/ Dependence of the Size of the Initially Collapsed Form During the Refolding of Barstar on Denaturant Concentration: Evidence for a Continuous Transition Kalyan K. Sinha and Jayant B. Udgaonkar Journal of Molecular Biology Volume 353, Issue 3, 28 October 2005, Pages 704-718

8/ Synchrotron Radiation Effects on Catalytic Systems As Probed with a Combined In-Situ UV-Vis/XAFS Spectroscopic Setup J. Gerbrand Mesu, Ad M. J. van der Eerden, Frank M. F. de Groot, and Bert M. Weckhuysen J. Phys. Chem. B 2005, 109, 4042-4047

9/ Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods Kosuke Maki, Hong Cheng, Dimitry A. Dolgikh and Heinrich Roder Journal of Molecular Biology Volume 368, Issue 1, 20 April 2007, Pages 244-255

10/ Detection of a Hidden Folding Intermediate of the Third Domain of PDZ Hanqiao Feng, Ngoc-Diep Vu and Yawen Bai Journal of Molecular Biology Volume 346, Issue 1, 11 February 2005, Pages 345-353

11/ Reduced Global Cooperativity is a Common Feature Underlying the Amyloidogenicity of Pathogenic Lysozyme Mutations Mireille Dumoulin, Denis Canet, Alexander M. Last, Els Pardon, David B. Archer, Serge Muyldermans, Lode Wyns, Andre Matagne, Carol V. Robinson, Christina Redfield and Christopher M. Dobson Journal of Molecular Biology Volume 346, Issue 3, 25 February 2005, Pages 773-788

12/ Sequence Determinants of a Protein Folding Pathway Chiaki Nishimura, Michael A. Lietzow, H. Jane Dyson, Journal of Molecular Biology Volume 351, Issue 2, 12 August 2005, Pages 383-392

13/ Interaction between the Catalytic Site and the A-M3 Linker Stabilizes E2/E2P Conformational States of Na+,K+-ATPase* Mads Toustrup-Jensen and Bente Vilsen J. Biol. Chem., Vol. 280, Issue 11, 10210-10218, March 18, 2005

14/ Functional Comparison between Secretory Pathway Ca2+/Mn2+-ATPase (SPCA) 1 and Sarcoplasmic Reticulum Ca2+-ATPase (SERCA) 1 Isoforms by Steady-state and Transient Kinetic Analyses* Leonard Dode, Jens Peter Andersen, Luc Raeymaekers, Ludwig Missiaen, Bente Vilsen, and Frank Wuytack J. Biol. Chem., Vol. 280, Issue 47, 39124-39134, November 25, 2005

15/ Conversion of 5-aminolevulinate synthase into a more active enzyme by linking the two subunits: Spectroscopic and kinetic properties Junshun Zhang, Anton V. Cheltsov and Gloria C. Ferreira Protein Science 2005 14: 1190-1200 Protein Sci.

16/ A Time-resolved Investigation of Ribosomal Subunit Association Scott P. Hennelly, Ayman Antoun, Måns Ehrenberg, Claudio O. Gualerzi, William Knight, J. Stephen Lodmell and Walter E. Hill, JMB Volume 346, Issue 5, 11 March 2005, Pages 1243-1258

17/ Pre-steady-state kinetic analysis of riboflavin synthase using a pentacyclic reaction intermediate as substrate Boris Illarionov, Ilka Haase, Markus Fischer, Adelbert Bacher, Nicholas Schramek Biological Chemistry386, Issue: 2, February 2005 pp: 127-136

18/ Enhanced picture of protein-folding intermediates using organic solvents in H/D exchange and quench-flow experiments Chiaki Nishimura, H. Jane Dyson, and Peter E. Wright Proc Natl Acad Sci U S A. 2005 March 29; 102(13): 4765–4770.

19/ Mutation of Gly-94 in transmembrane segment M1 of Na+,K+-ATPase interferes with Na+ and K+ binding in E2P conformation Anja Pernille Einholm, Mads Toustrup-Jensen, Jens Peter Andersen, and Bente Vilsen Proc Natl Acad Sci U S A. 2005 August 9; 102(32): 11254–11259.

20/ Oxidation of heparan sulphate by hypochlorite: role of N-chloro derivatives and dichloramine-dependent fragmentation Martin D. REES, David I. PATTISON and Michael J. DAVIES1 Biochem. J. (2005) 391 (125–134)

21/ The electrochemical self-assembly of the FAD modified zinc oxide films and their electrocatalytic properties Kuo-Chiang Lin and Shen-Ming ChenCorresponding Author Contact Information, Journal of Electroanalytical Chemistry Volume 578, Issue 2, 1 May 2005, Pages 213-222

22/ Cooperative Mechanism of RNA Packaging Motor Jiri Lisal and Roman Tuma JBC Vol. 280, No. 24, Issue of June 17, pp. 23157–23164, 2005

23/ The 3'-Azido Group Is Not the Primary Determinant of 3'-Azido-3'-deoxythymidine (AZT) Responsible for the Excision Phenotype of AZT-resistant HIV-1 THE JOURNAL BIOLOGICAL CHEMISTRY Vol. 280, No. 32, Issue of August 12, pp. 29047–29052, (2005) Nicolas Sluis-Cremer, Dominique Arion, Urvi Parikh, Dianna Koontz, Raymond F. Schinazi, John W. Mellors, and Michael A. Parniak

24/ Protonation Sites of Indoles and Benzoylindoles Francisco J. Hoyuelos, Begoña García, Saturnino Ibeas, María S. Muñoz, Ana M. Navarro, Indalecio Peñacoba, José M. Leal European Journal of Organic Chemistry Volume 2005, Issue 6 , Pages 1161 - 1171

25/ Folding of the SARS Coronavirus Spike Glycoprotein Immunological Fragment (SARS•S1B): Thermodynamic and Kinetic Investigation Correlating with Three-Dimensional Structural Modeling† Changying Yu, Chunshan Gui, Haibin Luo, Lili Chen, Liang Zhang, Hao Yu, Sheng Yang, Weihong Jiang, Jianhua Shen, Xu Shen, and Hualiang Jiang Biochemistry 2005, 44, 1453-1463

26/ Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles Kaare Teilum, Thorsten Thormann, Nigel R. Caterer, Heidi I. Poulsen, Peter H. Jensen, Jens Knudsen, Birthe B. Kragelund, Flemming M. Poulsen Proteins: Structure, Function, and Bioinformatics, 2005, Volume 59, Issue 1 , Pages 80 - 90

27/Folding and particle assembly are disrupted by single-point mutations near the autocatalytic cleavage site of Nudaurelia capensis virus capsid protein Derek J. Taylor and John E. Johnson Protein Science (2005), 14:401-408

28/ Comparison of Microenvironments of Aqueous Sodium Dodecyl Sulfate Micelles in the Presence of Inorganic and Organic Salts: A Time-Resolved Fluorescence Anisotropy Approach G. B. Dutt Langmuir, 21 (23), 10391 -10397, 2005

29/ Mesures en temps réel par diffusion de neutrons aux petits angles (DNPA) avec un appareil à « flux stoppé » (stopped-flow) = Real time study by small angle neutron scaterring with a stopped flux equipment GRILLO I. Neutrons et biologie. Ecole Thématique, Praz-sur-Arly Journal de physique. IV 2005, vol. 130 , pp. 75-80

30/ Protein folding: defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins. Maxwell KL et al (2005) Protein Science 14, 602-616

31/ Probing fast kinetics in complex fluids by combined rapid mixing and small-angle X-ray scattering. P. Panine, S. Finet, T. Weiss, T. Narayanan Advances in Colloid and Interface Science, Volume 127, Issue 1, Pages 9-18

32/ Role of RhAG and AQP1 in NH3 and CO2 gas transport in red cell ghosts: a stopped-flow analysis P. Ripoche, D. Goossensa, O. Devuystd, P. Ganea, Y. Colina, , A.S. Verkmane and J.-P. Cartrona,, Transfusion Clinique et Biologique Volume 13, Issues 1-2, March-April 2006, Pages 117-122

33/ Snapshots of cytochrome c folding Ekaterina V. Pletneva, Harry B. Gray, and Jay R. Winkler PNAS December 20, 2005 vol. 102 no. 51, 18397–18402

34/ Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells Nedjma ZIDI-YAHIAOUI, Isabelle MOURO-CHANTELOUP, Anne-Marie D'AMBROSIO, Claude LOPEZ, Pierre GANE, Caroline LE VAN KIM, Jean-Pierre CARTRON, Yves COLIN1 and Pierre RIPOCHE Biochem. J. (2005) 391 (33–40)

35/ An advanced EPR stopped-flow apparatus based on a dielectric ring resonator Günter Lassmann, Peter Paul Schmidt and Wolfgang Lubitz Journal of Magnetic Resonance, Volume 172 Issue 2 , February 2005, Pages 312-323

36/ Stabilization of Native Protein fold by intein-mediated covalent cyclization N.Williams, E.liepinsh, S.Watt, P.Prosselkov, J.Matthews, P.Attard, J.Beck, N.Dixon, G.Otting L.Mol.Biol.(2005) 346, 1095-1108

37/ Pore selectivity analysis of an aquaglyceroporin by stopped-flow spectrophotometry on bacterial cell suspensions. Hubert JF, Duchesne L, Delamarche C, Vaysse A, Gueune H, Raguenes-Nicol Biol Cell. 2005 Sep;97(9):675-86. note

38/ RecQ Helicase-catalyzed DNA unwinding detected by fluorescence resonance energy transfer X-D.Zhang, S-X.Dou, P. Xie, P-Y Wang, X-G Xi Acta biochimica et Piophysica Sinica 2005, 37(9): 593-600. note

39/ Cooperative Sub-Millisecond Folding Kinetics of Apomyoglobin pH 4 Intermediate Sebastien Weisbuch, Francine Gérard, Marielle Pasdeloup, Jéremy Cappadoro, Yves Dupont, and Marc Jamin Biochemistry 2005, 44, 7013-7023.

40/ Reduction of ferric haemoproteins by tetrahydropterins: a kinetic study Chantal CAPEILLERE-BLANDIN, Delphine MATHIEU and Daniel MANSUY Biochem. J. (2005) 392, 583–587

41/ The Formin Homology 1 Domain Modulates the Actin Nucleation and Bundling Activity of Arabidopsis FORMIN1 Alphée Michelot, Christophe Guérin, Shanjin Huang, Mathieu Ingouff, Stéphane Richard, Natalia Rodiuc, Christopher J. Staiger, and Laurent Blanchoin Plant Cell. 2005 August; 17(8): 2296–2313

42/ pH-Induced Micellization Kinetics of ABC Triblock Copolymers Measured by Stopped-Flow Light Scattering Zhiyuan Zhu, Steven P. Armes, and Shiyong Liu Macromolecules 2005, 38, 9803-9812

43/ Trifluoroethanol-Induced Unfolding of Concanavalin A: Equilibrium and Time-Resolved Optical Spectroscopic Studies Qi Xu and Timothy A. Keiderling Biochemistry, 44 (22), 7976 -7987, 2005

44/ Stabilization of Native Protein fold by intein-mediated covalent cyclization N.Williams, E.liepinsh, S.Watt, P.Prosselkov, J.Matthews, P.Attard, J.Beck, N.Dixon, G.Otting L.Mol.Biol.(2005) 346, 1095-1108

45/ Pore selectivity analysis of an aquaglyceroporin by stopped-flow spectrophotometry on bacterial cell suspensions. Hubert JF, Duchesne L, Delamarche C, Vaysse A, Gueune H, Raguenes-Nicol C. Biol Cell. 2005 Sep;97(9):675-86. note

46/ Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes HOW WELL ARE PROTEIN FOLDING AND STABILITY ASPECTS COUPLED? Rajesh Mishra, Robert Seckler, and Rajiv Bhat THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 16, Issue of April 22, 2005 pp. 15553-15560

47/ Glucocorticoids increase osmotic water permeability (Pf) of neonatal rabbit renal brush border membrane vesicles Jaap Mulder, Sumana Chakravarty, Maha N. Haddad, Michel Baum, and Raymond Quigley Am J Physiol Regul Integr Comp Physiol 288: R1417-R1421, 2005

Also see our selection of scientific articles using the Bio-Logic rapid kinetics systems

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