Low Temperature Beaker
Stopped Flow Sample Handling

Application notes

The following application notes are available.

Please click on the relevant application note to see a synopsis.

If you would like to request an application note, please use our information request form.


Ref. AN.001.H56
Pressure Effects On Protein-Protein Interaction In Rabbit Muscle


Ref. AN.002.S20
Optimisation Of Rapid Kinetics Data From A Commercial Spectrofluorimeter Using A Stopped-Flow Accessory


Ref AN.003.S60
Peroxide Oxidation Of Mutant Cytochrome c Peroxidase


Ref. AN.004.S60
Catalytic Reactions Involving Parahydroxybenzoate Hydroxylase


Ref. AN.005.R63
Determination Of Rate Constants For The Sequential Cleavage Of Double Stranded DNA Using Quench Flow Methods


Ref. AN.006.S60
Stopped-flow Studies Using Conductimetric Monitoring.


Ref. AN.007.S20
Reaction Mechanisms in Calcium-Calmodulin-Peptide Complexes


Ref. AN.008.HS20
Stopped-Flow Kinetics of Ellman's Reagent with the Beckman DU7500 Diode Array Spectrophotometer


Ref. AN.010.S60
The Kinetics of Catalysed and Uncatalysed Dismutation of Superoxide using a novel approach to Multi-Solution Mixing


Ref. AN.011.S20
Rapid Kinetics study of the displacement of Calcium ions bound to FURA-2 using the SFA-20 Stopped-Flow accessory with a PTI Spectrofluorimeter


Ref. AN.012.S60
Binding Studies of a DNA Repair Enzyme using Stopped-Flow Fluorescence Anisotropy


Ref. AN.013.S60
ATP Binding to Rabbit Skeletal Subfragment 1 to reveal sensitivity of the Hi-Tech KinetAsyst™ stopped-flow system


Ref. AN.014.S20
Measurement of the Reduction of Potassium Ferricyanide by L-Absorbic Acid with a Shimadzu UV-1700 Spectrophotometer Using a Stopped-Flow Accessory


Ref. AN.015.S20
Measuring the formation of 2,4-Dinitrophenol from 2,4-Dinitrophenol Acetate and Sodium Hydroxide using a Varian Cary 50 Spectrophotometer with Stopped-Flow Accessory


Ref. AN.017.S20

Measure the displacement of a Mant Nucleotide from a complex with S1 using a Varian Eclipse Fluorimeter with a Stopped-Flow accessory


Ref. AN.018.S20

Fluorescent measurements using a Stopped-Flow accessory with an ISS PC1 Fluorimeter


Ref. AN.019.T64
Temperature calibration of T-Jump Instrument TJ-64 using Tris/Phenol Red


Ref. AN.020.T64
Ionic Strength Dependence of the Heating Time in T-Jump Instrument TJ-64

 

Other Manufacturers' Application Notes


Ref. AN.HP8452A
Stopped-flow kinetics with the Hewlett Packard HP8452A UV/Vis
Spectrophotometer and SFA-20


Ref. AN.HP8453
Fluorescence reaction kinetics using the stopped-flow technique
(Hitachi F-4500) using an optimised SFA-20


Ref. FL.B.3
Fluorescence reaction kinetics using the stopped-flow technique (Hitachi F-4500) using an optimised SFA-20


Ref. UV.75
Monitoring fast reactions using stopped-flow kinetics on the Cary 50 and Cary 300 using an optimised SFA-20.

Synopsis

Application Note - AN.001.H56

Variable pressure Stopped-flow studies of the binding of myosin subfragment S1 to actin molecules from rabbit muscle

A dynamic interaction between the proteins in thick myosin and thin actin filaments in muscle is believed to generate mechanical force in muscle contraction¹

Keywords

  1. High pressure stopped-flow
  2. Turbidity
  3. Muscle-protein

References

  1. ¹M. A. Geeves Biochem. J. (1991) 274, 1-14
  2. ²L. E. Greene & E. Eisenberg, Proc. Natl. Acad. Sci. U. S. A. (1980) 77, 2616-2620
  3. ³M. A. Geeves & D. J. Halsall, Proc. R. Soc. London Ser. B. (1986) 229, 85-95
TgK Scientific Ltd would like to thank Dr. M. A. Geeves of the Max Planck Institute of Molecular Physiology, Dortmund, Germany for the work reported here.

Application Note - AN.002.S20

Evaluation of rapid kinetics data from a vertical beam commercial spectrofluorimeter using an optimised SFA-20 stopped-flow accessory

The range of kinetic reactions that can be studied by commercial absorbance and fluorescence spectrophotometers can be greatly enhanced by the addition of a stopped-flow accessory. A study of the rate of reaction between N-acetyltryptophanamide and N-bromosuccinimide has been used to evaluate the performance of a spectrofluorimeter with a vertical beam geometry using an optimised version of the stopped-flow accessory.

Keywords

  1. Stopped-flow accessory
  2. Perkin-Elmer LS-50B
  3. Spectrofluorimeter
  4. Optimised geometry

Application Note - AN.003.S60

Low absorbance studies of peroxide oxidation of mutant cytochrome c peroxidase using diode array stopped-flow techniques

The diode array stopped-flow technique offers a powerful method of aquiring multi-wavelength data from rapid reactions from a single shot. The oxidation of a mutant cytochrome c peroxidase by hydrogen peroxide is used to show how data can be readily obtained from fast reactions that exhibit only small absorbance changes. Full data analysis is then carried out using SPECFIT, a multi-variate global analysis software package.

Keywords

  1. Diode array stopped-flow
  2. cytochrome c peroxidase
  3. multi-variate global analysis

References

  1. ¹Biochemistry, 1989, 28, 7992, Erman et al.
TgK Scientific Ltd would like to thank Dr.J. Erman, Northern Illinois University, USA, for the supply of CcP(MI, F191) solution.

Application Note - AN.004.S60

Identification of short-lived intermediates in Parahydroxybenzoate Hydroxylase mechanisms by stopped-flow techniques and global analysis.

The combination of diode array stopped-flow spectrophotometry and multi-variate global analysis can be used to identify short-lived intermediates and hence reaction mechanisms from fast reactions. Studies of catalytic reactions involving the enzyme parahydroxybenzoate hydroxylase provide a powerful illustration of the capabilities of such a combination.

Keywords

  1. Singular Value Decomposition
  2. short-lived intermediates
  3. diode-array stopped-flow
  4. parahydroxybenzoate hydroxylase
  5. multi-variate global analysis

References

  1. ¹SPECFIT, Spectrum Software Associates, P. O. Box 4494, Chapel Hill, NC 27515-4494
  2. ²B. Entsch and D. Ballou, (1989), Biochim. Biophys. Acta, 999, 313-322
TgK Scientific Ltd would like to thank Dr. Palfrey, Prof. D Ballou and Mr. E. J. King who carried out the work reported here.

Application Note - AN.005.R63

Rapid quench-flow studies of enzymatic cleavage of covalently closed circular DNA

The rapid quench flow technique provides a powerful method of studying rapid reactions involving solutions that are not chromophores and so cannot be monitored by absorbance or fluorescence spectrophotometry, or are at concentrations too low to allow the use of an optical probe. This is illustrated by studies of the cleavage of the plasmid pAT153, a covalently closed circle of DNA, by the restriction enzyme EcoRV.

Keywords

  1. Rapid quench flow
  2. restriction enzyme
  3. recognition sequences
  4. EcoRV

References

  1. ¹Fritz K. Winkler and Dirk Kostrewa, Biochemistry 1995, 34, 683-696
TgK Scientific Ltd would like to thank Dr. S. E. Halford and S. G. Erskine, Department of Biochemistry, Centre for Molecular Recognition, University of Bristol, UK, for the work reported here.

Application Note - AN.006.S60

Conductimetric monitoring of proton loss from acetylacetone in excess dilute alkali

Absorbance and fluorescence spectrophotometry are the usual techniques used to follow the progress of a reaction in a stopped-flow cell. However, not all reactions produce a measurable change in the spectrum and conductimetric detection methods can provide a useful alternative. The use of the conductivity method is illustrated here by monitoring the loss of a proton from acetylacetone in the presence of dilute alkali.

Keywords

  1. Conductivity
  2. Stopped-flow
  3. conductimetric monitoring

References

  1. ¹J. E. Crooks. Hosny El-Daly, Mohammed Y El-Sheikh, Abdul-Fattah M Habib and Ahmed B Zaki, Int. J. Chemical Kinetics, 25, 1993, 161-168.
TgK Scientific Ltd would like to thank Dr. John Crooks, Department of Pharmacy, King's College, London, for the measurements reported here.

Application Note - AN.007.S20

Dissociation kinetics of calcium-calmodulin and calcium-calmodulin-peptide complex using the SFA-20 stopped-flow fluorescence accessory

Calcium-saturated calmodulin (Ca4-CaM) is involved in the regulation of a wide range of cellular functions. Dissociation of calcium from Ca4-CaM, and from complexes formed between Ca4-CaM and target peptides, frequently occurs in two kinetically distinct steps. The second (slowest) step, associated with the loss of two C-terminal calcium ions, can be studied by stopped-flow methods using a commercial spectrofluorimeter equipped with an SFA-20 stopped-flow accessory.

Keywords

  1. Calmodulin
  2. target peptides
  3. stopped-flow accessory
  4. SPEX FluoroMax
  5. spectrofluorimeter

References

  1. ¹S. R Martin, J. F. Maune, K. Beckingham and P. M Bayley (1992) Eur. J. Biochem. 205:1107-1114
  2. ²W. A. Findlay, S. R. Martin, K. Beckingham and P. M. Bayley, Biochemistry, (1995), 34:2087-2094.
TgK Scientific Ltd would like to thank Dr. S. R. Martin, National Institute for Medical Research, London, for the work reported here.

Application Note - AN.008.HS20

Stopped-flow kinetics of Ellman's reagent with thioglycerol using the SFA-20 stopped-flow accessory optimised for use with the Beckman DU 7500 Diode array spectrophotometer

The use of a commercial spectrophotometer equipped with a diode array detector to study rapid reaction kinetics has great potential due to the simultaneous acquisition of multi-wavelength data. This is made possible by the addition of an optimised stopped-flow accessory. The reaction of Ellman's reagent with thioglycerol is a well characterised coupled two step process and is used to illustrate how this combination can be used to acquire complex data which can then be processed using multi-variate global analysis methods.

Keywords

  1. stopped-flow accessory
  2. Beckman DU 7500
  3. diode array
  4. multi-variate global analysis

References

  1. ¹Carlton Paul, Kasper Kirschner and Gernot Haenish, Analytical Biochemistry, 101, 442-448 (1980).
TgK Scientific Ltd would like to thank Tim Ward at Beckman UK for access to the DU7500.

Application Note - AN.010.S60

The kinetics of catalysed and uncatalysed dismutation of superoxide using a novel approach to multi-solution mixing in a computer-controlled stopped-flow system.

The use of multi-mixing techniques is well established in stopped-flow studies of rapid reactions. However, the introduction of an extended push plate enabling all syringes to be driven simultaneously has greatly facilitated the study of "difficult" reactions. These include applications where the mixing of different solvents may produce unwanted artefacts, or where large dilutions between solutions of different densities are required, such as in protein folding. The use of such a system for the study of the catalysed and uncatalysed dismutation of superoxide is described here.

Keywords

  1. Dismutation
  2. superoxide
  3. computer controlled stopped-flow
  4. diode array
  5. novel multi-mixing

References

  1. ¹J. A. Fee and J. S. Valentine, "Chemical and Physical Properties of Superoxide" in Superoxide and Superoxide Dismutases, A. M. Michelson, J. M. McCord and I. Fridovich, eds., (1977) Academic Press, London, New York, San Francisco.
  2. ²Mc Clune, G. J. and Fee, J. A. (1976) FEBS Letters, 67, 294-298
TgK Scientific Ltd would like to thank Dr. Chris Bull and Prof. Dave Ballou (University of Michigan) for their help and support.

Application Note - AN.011.S20

Rapid kinetics study of the displacement of calcium ions bound to FURA-2 using the SFA-20 stopped-flow accessory with a PTI spectrofluorimeter

Measurement of changes in concentration of intracellular cations such as Ca²+ is important in understanding the mechanisms of many cellular processes. The use of a fluorescence indicator in combination with fluorescence spectrophotometry is a well established technique for making these measurements. The rate of displacement of the fluorescence indicator from the bound complex is important to ensure the validity of quantitative results. The rate of this displacement can be measured using a commercial spectrofluorimeter equipped with a stopped-flow accessory.

Keywords

  1. SFA-20 Stopped-flow accessory
  2. PTI QuantaMaster C-60
  3. Intracellular calcium ion
  4. Fura-2

References

  1. ¹Jackson, A.P., Timmerman, M.P., Bagshaw, C.R. and Ashley, C.C. (1987 FEBS Letters 216, 35-39
  2. ²G. Grynkiewicz, M. Peonie, and R.Y. Tsien, Journal of Biological Chemistry, 260, (1985), No. 6

Application Note - AN.012.S60

Binding studies of a DNA repair enzyme using stopped-flow fluorescence anisotropy

The use of fluorescence stopped-flow methods for the elucidation of rapid kinetics in solution is well characterised. By using a plane polarised excitation beam, however, the intensity of fluorescence in directions parallel and perpendicular to the direction of polarisation can be measured. These two components can be combined so as to express a convenient technique to investigate the binding of a protein to DNA. This technique uses an oligonucleotide that is labelled during synthesis with a commercially available fluorescent dye. It can thus be applied to any DNA binding protein and does not rely on any intrinsic fluorescence properties of the protein. This application note reports on the binding of a DNA repair enzyme, uracil DNA glycosylase, to an oligonucleotide containing uracil.

Keywords

  1. Fluorescence polarisation
  2. stopped-flow
  3. anisotropy
  4. DNA-protein interactions
TgK Scientific Ltd would like to thank Dr G Baldwin, Dept of Biochemistry, University of Bristol, Bristol, UK for the work reported here.

Application Note - AN.013.S60

ATP Binding to Rabbit Skeletal Subfragment 1 to reveal sensitivity of the Hi-Tech KinetAsyst™ stopped-flow system

Muscle movement is believed to be caused by the generation of force produced by the cyclic interaction of the thick myosin and the thin actin filaments in muscle. Here we report on the sensitivity of the Hi-Tech KinetAsyst™ stopped-flow system by following the binding of ATP to rabbit skeletal muscle myosin under conditions where the protein concentration is lowered until a signal is no longer measurable

Keywords

  1. Fluorescence
  2. sensitivity
  3. stopped-flow
  4. KinetAsyst™
TgK Scientific Ltd would like to thank Prof. M. A. Geeves and Mr R. Clark from the University of Kent, Canterbury, UK, for the work reported here.

Application Note - AN.014.S20

Measurement of the Reduction of Potassium Ferricyanide by L-Absorbic Acid with a Shimadzu UV-1700 Spectrophotometer Using a Stopped-Flow Accessory

The reduction of Potassium Ferricyanide by Absorbic Acid is a well-known kinetic reaction that was first published by Tonomura et al (Analytical Biochemistry 84 (1978) pp 370-383). The speed of this reaction is dependent on the pH value of the solution which makes this reaction a very useful one for testing the performance of kinetic instruments.

Keywords

  1. stopped-flow Accessory
  2. Shimadzu UV-1700
  3. Rapid kinetics
TgK Scientific Ltd would like to thank Shimadzu U.K for making the UV-1700 available to us for measurements.

Application Note - AN.015.S20

Measuring the formation of 2,4-Dinitrophenol from 2,4-Dinitrophenol Acetate and Sodium Hydroxide using a Varian Cary 50 Spectrophotometer with Stopped-Flow Accessory

The hydrolysis of 2,4-Dinitrophenol Acetate by Sodium Hydroxide has become a standard reaction for testing the performance of fast kinetic instruments. The reaction was first published by H Gutfreund (Methods in Enzymology 16 (1969) pp 229-249) and can be measured with a Varian Cary 50 Spectrophotometer and SFA-20 stopped-flow accessory because of the very fast time-base this spectrophotometer has.

Keywords

  1. stopped-flow Accessory
  2. Varian Cary 50
  3. Rapid kinetics
  4. SFA-20

Application Note - AN.017.S20

Measure the displacement of a Mant Nucleotide from a complex with S1 using a Varian Eclipse Fluorimeter with a Stopped-Flow accessory

The mechanism of the ATPase reaction of myosin and its subfragment, S1 has been studied with transient kinetic methods and is widely reported. Here, a derivative of ADP, mantADP [2'-3'-O-(N'-methylanthraniloyl-5'-O-diphosphate] in premixed complex has been displaced with excess ATP (Woodward, Eccleston and Geeves, 1991). This demonstrates the use of the SFA-20 rapid mixing accessory (Hi-Tech Scientific), in a commercial fluorescence instrument.

Keywords

  1. Stopped-Flow Accessory
  2. Varian Eclipse
  3. Rapid Kinetics
  4. SFA-20
  5. Fluorescence

Application Note - AN.018.S20

Fluorescent measurements using a Stopped-Flow accessory with an ISS PC1 Fluorimeter

N-acetyltryptophanamide (NATA) and N-bromosuccinimide (NBS) as used by Peterman and additionally the displacement of calcium from Drosophila calmodulin both demonstrate the use and quality of data that can be achieved with the SFA-20 rapid mixing accessory with an ISS PC1 Fluorimeter.

Keywords

  1. Stopped-Flow Accessory
  2. SFA-20
  3. ISS PC1
  4. Rapid Kinetics
  5. Tryptophan
  6. Tyrosine
  7. Fluorescence
  8. Dead Time
  9. Calmodulin

Application Note - AN.019.T64

Temperature calibration of T-Jump Instrument TJ-64 using Tris/Phenol Red

The Hi-Tech Scientific T-Jump instrument TJ-64 enables the experimentalist to vary the magnitude of the temperature jump by changing the discharge voltage. This in turn allows ‘tuning’ of the magnitude of the equilibrium perturbation and consequently the extent of signal change. In order to calibrate the observed temperature changes corresponding to the applied discharge voltages, the coupled reaction of Tris/phenol red was examined using relaxation amplitude analysis.

Keywords

  1. T-Jump
  2. TJ-64
  3. Temperature calibration
  4. Phenol red
  5. Tris

Application Note - AN.020.T64

Ionic Strength Dependence of the Heating Time in T-Jump Instrument using Fluorescence

This application note shows that the observed rate of heating (kobs) depends on the ionic strength of the NATA solution.

Keywords

  1. T-Jump
  2. TJ-64
  3. N-Acetyl-L-tryptophanamide
  4. Fluorescence
  5. Heating time
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