Module 4 isolation of chloroplasts and characterization of photosynthetic pigment 2
- 1. ISOLATION OF CHLOROPLASTS AND CHARACTERIZATION OF PHOTOSYNTHETIC PIGMENT CMB Lab. Activity 4 Ms. O.Hara
- 2. Studying subcellular components ◦ Composition – the native structure of the cell organelles ◦ Function – the activity of their components ◦ Morphological ◦ Biochemical where component structures ◦ Biophysical difffer ◦ Physiological
- 3. Studying subcellular components ◦ usually initiated by cell disruption ◦ To receive a mixture of organelles which is very convenient for further separation.
- 4. Cell fractionation ◦ The resulting homogenate is composed of the broken cells with subcellular components released in a buffered medium. ◦ Debris (unseparated and unbroken cells) must be separated by standing the homogenate for some time or by filtration. ◦ Sediment or the residue is discarded ◦ Supernate or the filtrate is subjected to CENTRIFUGATION.
- 5. Centrifugation ◦ a process that involves the use of the centrifugal force for the separation of mixtures used in laboratory, increasing the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate (pellet) to gather on the bottom of the tube. The solution (supernatant) is then either decanted or used in other step. The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in RPM or g.
- 6. Centrifugation ◦ used for the isolation and purification of organelles and macromolecules. ◦ when a particle is subjected to centrifugal force by spinning a cellular extract at extremely rapid rates in a laboratory centrifuge, the rate of movement of the particle through a specific solution depends on its size and density, as well as the solution’s density and viscosity.
- 7. Types of Centrifuges • Low-speed centrifuge - table centrifuge - typical maximum speed is 6000rpm. - room temp. - cell, nucleus, etc. (easily precipitated material) • High-speed centrifuge - max. speed 20000-25000rpm (60000 X g) - temp. control - cell, nucleus, organelle, etc. • Ultracentrifuge - max. speed 9800km/s2 (1000000 X g) - temp. control and vacuum system - cell, nucleus, organelle, components of memb., polysome, macromolecule, etc.
- 8. Types of centrifugation ◦ Differential centrifugation is a procedure in which the homogenate is subjected to repeated centrifugations each time increasing the centrifugal force.
- 9. Types of centrifugation ◦ Density gradient centrifugation is a procedure for separating particles in which a sample is placed in a preformed gradient such as sucrose. Upon centrifugation, the particles are “banded” in the gradient and can be collected as a pure fraction. Example:
- 10. Paper Chromatography ◦ Paper chromatography is a technique that can separate compounds on paper by a solvent system that carries the mixture of pigments up the paper by capillary action. ◦ Two phases are formed: ◦ the mobile phase - solvent ◦ stationary - chromatography paper ◦ separation is caused by a difference in distribution of the components between the stationary phase and the mobile phase
- 11. Spectrophotometry ◦ Spectrophotometer - an instrument which measures the absorption or transmission of light by a solution at any wavelength(s) selected by the experimenter.
- 12. Part 1: Isolation of Chloroplast ◦ http://vlab.amrita.edu/?sub=3&brch=187&sim=878&cnt=2 ◦ https://youtu.be/6JJBvh-NQZA
- 13. Isolation of Chloroplasts from a plant sample Leaves of spinach, lettuce are commonly used for the isolation of chloroplasts. Intact chloroplasts are the best source for studying the processes like carbon assimilation, electron flow and phosphorylation.
- 14. Estimation of chlorophyll concentration 1. Add 10 ul of chloroplast suspension to 990ul of 80% acetone solution and mix gently. 2. Centrifuge at 3000xg for 2 minutes. 3. Take 100ul of the supernatant and transfer into a cuvette and measure the absorbance at 650 nm. Use 100 ul of 80% acetone as blank. 4. Take duplicate OD 650 values. 5. Take the average of the two values and estimate the mg/ml chlorophyll concentration using the following formula: Where A 650 is the absorbance at 650 nm, 100 is the dilution factor and 36 is the extinction coefficient of chlorophyll. http://cbi-au.vlabs.ac.in/cell-biology-1/Isolation_of_Chloroplast/experiment.html A 650 x 100/36 = mg/ml chlorophyll.
- 15. Worksheet 4a – Isolation of Chloroplasts ◦ A. Schematic Diagram ◦ B. Results ◦ Microscopy. ◦ chlorophyll concentration (mg/ml) of the chloroplast isolates ◦ Answers to guide questions
- 16. Part 2. Photosynthetic Pigments ◦ https://www.youtube.com/watch?v=FQqVqVZgKjA
- 17. • With a capillary tube, the mixture is streaked on the chromatography paper: enough sample is applied so that there will be an adequate amount for subsequent extraction and spectrophotometric analysis.
- 18. B. Separation of Photosynthetic Pigments ◦ chromatography strip (punctured on top) ◦ Crude chlorophyll suspension applied on the marked area (1.5 cm from the bottom) ◦ Lower the bottom edge with the extract towards the developing solution. ◦ Allow to develop
- 19. Characterization of photosynthetic Pigments by Rf value ◦ Rf – Reference front 1. Remove the developed chromatogram (separated pigments) from the developing chamber. 2. Quickly before it dries, mark where the solvent stopped with a pencil. This is called the solvent front. 3. Also mark where each pigment stopped moving. 4. Measure the distance traveled by the pigment from the origin and distance travelled by the solvent (developing solution) from the origin. Rf = distance pigment migrated distance solvent front migrated What is the Rf value for carotene calculated from the chromatogram?
- 20. Characterization of photosynthetic Pigments by absorbance spectrum ◦ The light absorbing photosynthetic pigments do not absorb all wavelengths of light equally. ◦ Plant pigments have specific wavelength absorbance patterns known as the absorbance spectrum. ◦ This will help determine which light waves are absorbed by the leaf pigments.
- 21. Developed chromatogram Yellow-orange Bluish green Yellow green Yellow
- 22. C. Elution of pigments ◦ Cut bands from strips ◦ Group according to color ◦ Put 1 group of colored band in a tube containing 4 ml of pure acetone. ◦ Shake for 5 -10 mins. ◦ Partially purified pigments ELUENTS
- 23. D. Spectrophotometry ◦ Place the blank containing pure acetone in a cuvette. ◦ Decant the eluents in individual cuvettes. ◦ Read the absorbance of each sample from 350 to 700 nm at 25 nm interval. ◦ Plot absorbance against wavelength. ◦ Compare the obtained spectra with the known spectra of the colored pigments found in reference books. ◦ Identify the pigments separated.
- 24. Absorbance Readings:Spinach leaf extract Wavelength (nm) ABSORBANCE Pigment 1 Pigment 2 Pigment 3 Pigment 4 360 0.032 0.065 0.015 0.009 380 0.033 0.085 0.017 0.011 400 0.035 0.091 0.025 0.018 420 0.047 0.113 0.035 0.026 440 0.064 0.106 0.042 0.033 460 0.067 0.021 0.038 0.037 480 0.037 0.015 0.036 0.034 500 0.006 0.008 0.007 0.015 520 0.005 0.008 0.002 0.004 540 0.007 0.009 0.001 0.002 560 0.006 0.011 0.002 0.0 580 0.009 0.017 0.003 0.001 600 0.011 0.019 0.001 0.001 620 0.010 0.026 0.0 0.0 640 0.018 0.029 0.001 0.0 660 0.030 0.083 0.0 0.0 680 0.009 0.034 0.001 0.0 700 0.001 0.003 0.0 0.0
- 25. Reference for Absorption spectra of plant photosynthetic pigments in acetone (Bilodeau et al. 2019) • Take note of the peaks or highest absorption values per pigment
- 26. ◦ A. Schematic diagram ◦ B. Results ◦ Give the Rf value of each separated pigment based on the chromatogram result. ◦ Make a line graph that shows the absorbance of each pigment at 360-700 nm. Use color pencils to represent each pigment. ◦ Compare the obtained spectra with the known spectra of the colored pigments found in reference books. What are the possible identities of the unknown pigments? ◦ Answers to guide questions Worksheet 4b