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LAB MANUAL-Analytical Techniques in Biotechnology BT-202
EXPERIMENT - 1
Objective: To study the principle and operation of Spectrophotometer. Principle: Spectroscopy: It is the measurement and interpretation of electromagnetic radiation absorbed, scattered, or emitted by atoms, molecules, or other chemical species. This absorption or emission is associated with changes in the energy states of the interacting chemical species and, since each species has characteristic energy states, spectroscopy can be used to identify the interacting species. Spectrophotometer: The instruments that are used to study the absorption or emission of electromagnetic radiation as a function of wavelength are called spectrometer or spectrophotometer. A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes. There are two major classes of spectrophotometers; single beam and double beam. A double beam spectrophotometer measures the ratio of the light intensity on two different light paths, and a single beam spectrophotometer measures the absolute light intensity. The most common spectrophotometers are used in the UV and visible regions of the spectrum, and some of these instruments also operate into the near- infrared region as well. Instrumentation: The starting point is the light source. Depending on the wavelength of interest, this can be an electrically powered ultraviolet, visible, or infrared lamp. The spectrophotometer's monochromator which selects the analytical wavelength from the source lamp's broad spectrum containing many wavelengths of light. The analytical wavelength is chosen based on the absorbance characteristics of the analyte. Monochromators are instruments whose sole purpose is to allow polychromatic (that is many wavelength containing) light into the entrance slit of the monochromator and only allow a single (or at least very few) wavelength (monochromatic light) out via the exit slit. In the instrumental design shown schematically the source lamp's beam is alternately diverted at right angles by a rotating disk with three distinct panels. One sector allows the beam to pass straight through the disk, another has a mirror surface, and a third is black. When the beam passes though the disk it shines directly into the sample cell. This cell contains a cuvette which is made of a transparent material, such as quartz, that does not absorb light in the spectral region of interest. The analyte is dissolved in a solvent held in the cuvette. When the source light is reflected at 90 degrees by the rotating disk instead of striking the sample cuvette it passes through a cuvette in the reference cell which contains only solvent.
SPSU text] [Type UDAIPUR Department of Biotechnology
LAB MANUAL-Analytical Techniques in Biotechnology BT-202
During the third sequence, when the black sector blocks the source beam, no light passes through the disk. And as can be seen below, therefore no light arrives at the phototranducer. This part of the cycle is used for the computer to digitize and measure the dark current - the amount of light produced by the phototransducer circuit when no light impinges on the phototransducer. The dark current can be subtracted from the overall light measurements made by the system. After travelling through either the sample cell or reference cell the light that was not absorbed--by far, most of the beam-- is directed onto the phototransducer or light detector. This component converts the arrival of photons into an electrical signal. The alternating light signals, from either the reference beam or sample beam generate alternating electrical phototransducer signals. A computer, sampling those signals, can now determine the analyte absorption in two ways. Some instruments merely subtract the sample beam signal's digitized light intensity from that of the reference beam. The difference is a measure of the amount of light absorbed by the analyte. Instrument required: 1. UV-Vis Spectrophotometer
Procedure: How to operate Double Beam Spectrophotometer 2203: 1. Switch on the instrument and wait till it finishes with the self calibration. 2. Refer the instruction manual for standardization if necessary (Should be done only after any position change of instruments or after servicing). 3. Give an initial warm up time of 30 min. 4. Carryout baseline calibration if required. Baseline calibration should be done periodically that will take care of any changes in baseline. Many factors can affect baseline like change and long time effect in electronics. 5. Once the baseline calibration is over set the required functions using the function keys and proceed further for the experiment.
the molecules may dimerize or aggregate which causes positive or negative deviation.D. When high sample concentrations are being measured. 3 .log10 I Absorbance shares linear relationship with sample concentration. Beer’s Law: The amount of light absorbed by a material is proportional to the number of absorbing molecules. Stating the Beer-Lambert law differently. Principle: Laws of absorbance of light: Lambert’s Law: The amount of light absorbed is proportional to the thickness of the absorbing material and is independent of the intensity of the incident light. 2. 5. Place 16 clean dry test tubes in test tube stand and label them 1 – 16. 2 to 15). i. Procedure: 1. and the reverse I/I0 is known as Transmittance. 6. Take 5ml of Q3 water in tube no. the quantity I0/I is known as O. Working DNA solution (500μg/ml): Dilute 10ml of stock solution to 100ml. Mix the contents. 3.e. 2. it can be said that optical density is proportional to the sample concentration if the path-length is constant. Stock DNA solution: Dissolve 50mg of DNA in 10ml of Q3 water to get a final concentration of 5mg/ml. 150 μl and respectively increasing amount upto 800 μl in other tubes (tube no. Beer-Lambert Law: The amount of light absorbed is proportional to the concentration of the absorbing substance and to the thickness of the absorbing material (Path length). Take cuvette and hold it on rough sides. the concentration of the absorbing solution. Materials required: 1. 1. A = log10 I0 . 4. Take 100 μl of working DNA in tube no. 16. Make up the volume to 5ml using Q3 water. Considering intensity of transmitted light as I and that of incident light as I0.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT: 2 Objective: To Verify the Beer-Lamberts Law using UV-Visible spectrophotometer.
3 70 14 750 4. Place it in the blank cell.0 0.7 5 25 5 300 4. 12. Cover the lid of the spectrophotometer and read the absorbance at 260nm using spectrophotometer. 11. Clean the cuvette with distilled water and rinse with small amount of Q3 water (Blank). D.45 55 11 600 4.2 80 16 Blank 5. μg/ml O.7 30 6 350 4.6 40 8 450 4. Dispense the blank solution in cuvette wipe all the four walls with tissue paper. Note down the O. clean it and rinse with small aliquote of sample. Take another cuvette for sample.9 10 2 150 4.4 60 12 650 4. 10.85 15 3 200 4. (260nm) 1 100 4. Working DNA solution (μl) Distilled water (ml) Final conc.25 75 15 800 4. Protocol: Name of the student: Test Tube No. 13.55 45 9 500 4. Dispense the sample in cuvette and place it in sample cell.8 20 4 250 4. 8.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 7. 4 . 9.35 65 13 700 4.00 Result/Interpretation: From the graph verify the Beer Lambert Law.65 35 7 400 4.5 50 10 550 4. with respect to the concentrations and plot the graph with the readings against the concentration.D.
It fluoresces under UV light when intercalated into DNA (or RNA). or RNA molecules by size. e. usually abbreviated as EtBr. Shorter molecules move faster and migrate farther than longer ones. Separation of restricted genomic DNA prior to Southern transfer. The technique is used for estimation of the size of DNA molecules following restriction enzyme digestion. By running DNA through an EtBr-treated gel and visualizing it with UV light. in restriction mapping of cloned DNA. Material Required: Agarose Gel rack or gel plate Comb Electrophoresis tank Electrodes Power Supply Gel documentation system Nitrile rubber gloves Weighing balance Ethidium bromide (10 mg/ml in H2O) Sample loading dye Composition: 1. e.g. or of RNA prior to Northern transfer.g. Principle: Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA. The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide. analysis of PCR products. Bromophenol blue 25mg 5 . This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT .3 Objective: To perform Submerged Agarose Gel Electrophoresis for Separation and Detection of Nucleic Acids. any band containing more than ~20ng DNA becomes distinctly visible. in molecular genetic diagnosis or genetic fingerprinting.
about 5-10 mm from the end of the gel.5 μg/ml.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 2.5g of Agarose powder in a 250ml conical flask. Distilled water 25mg 4g 10ml Buffer solution: TAE 1. or water bath. 1. Stir or swirl the solution while cooling. Sucrose 4. pH 8. 4. Mix it with 150 ml 1x TAE buffer and then heat in a microwave oven or on hot plate until completely melted. Wear gloves from here on.0 Composition of stock TAE 50x (pH 8. Then pour it into the gel rack. Add 5 µl ethidium bromide stock (10 mg/ml) per 100 ml gel solution for to the gel at a final concentration of 0. together with the rack. 6. 2. When the gel has cooled down and become solid. 6 . 7. 5.1 ml 200 ml 1000 ml Preparation of 1x TAE Buffer: TAE Buffer stock (50x) Distilled water 2ml 100ml Procedure: Prepare 1% agarose gel: Weigh 1. into electrophoretic tank containing 1x TAE.0): Tris Base Glacial Acetic acid EDTA (0. Insert the comb at one side of the gel.0x. 3. ethidium bromide is a mutagen. Mount the gel. Stir the solution to disperse the ethidium bromide. Xylene cyanol 3. Let the solution cool down to about 50 °C at room temperature. The holes that remain in the gel are the wells or slots.25M) Volume make up to 242 g 57. Be very careful when handling the concentrated stock. carefully remove the comb.
14. DNA will migrate towards the positive electrode (anode) which is usually colored red. with the slots at the end electrode that will have the negative current. 9. Examine the gel on UV transilluminator and document with the gel documentation system. The gel must be completely covered with TAE. 7 . Confirm the flow of current by observing bubbles coming off the electrodes. 11. Mix the sample of DNA with 0. Place the cover over the tank and connect the electrode wires at the respective positions in the electrophoretic tank and to the power pack. 15. Result/ Interpretation: Report the result in terms of separation and detection analysis. When the DNA samples or dyes have migrated a sufficient distance through the gel. 13.2 volume of the 6x gel-loading buffer (eg.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 8. 12. 10. Observation: Report the observation from the image obtained after documentation. turn off the electric current and remove the electrodes and cover from the gel tank. Mix 5μl DNA sample with 1μl gel loading buffer). Apply 50V current and set the timer for 2h. 16. Load the DNA molecular weight marker (DNA Ladder) mixed with loading dye beside the sample well. Load the DNA samples mixed with loading buffer slowly into the sample wells using micropipette.
4.1mg/ml) NADH (0. The pattern of energy absorption by a substance when light of varying wavelength passes through it is uniquely characteristic of substance. 2. 8 . Dispense the blank solution in cuvette wipe all the four walls with tissue paper.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT .4 Objective: To Determination of absorption maxima (λmax) of the given sample by UV-Visible spectrophotometer. Clean the cuvette with distilled water and rinse with small amount of blank sample (MiliQ water). Principle: When sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule. The wavelength at which the compound absorbs most is known as its absorption maxima. 5. some of the light energy will be absorbed as the electron is promoted to a higher energy orbital. Absorbance usually ranges from 0 (no absorption) to 2 (99% absorption). Take cuvette and hold it on rough sides. 3. Place it in the blank cell. 6.1mg/ml) UV-Vis Spectrophotometer Tissue paper Cuvettes MiliQ Water Procedure: 1. The resulting spectrum is presented as a graph of absorbance (A) versus wavelength. Switch on the spectrophotometer and give a warm up time of 30min. Prepare the amino acid solutions in MiliQ water.1mg/ml) Bromophenol blue solution (0. Materials Required: Amino acid (Lysine. This pattern is known as absorption spectrum. Phenylalanine) solutions in MiliQ water (0. and is precisely defined in context with spectrometer operation.1mg/ml) Bovine serum albumin (0.
clean it and rinse with small aliquote of sample. Switch on the UV and Vis lights and select the multi wavelength option from the main menu in spectrophotometer. 11. Data Table: Wavelength () Absorbance Wavelength () Absorbance Result/Interpretation: Determine the peak on the graph and report the absorption maxima for the given amino acid. Take another cuvette for sample. Observation: Report the observation in following data table and plot the graph using the absorbance values against the wavelength. 9. For Bromophenol blue solution read the absorbance in the range of 300 – 700nm. 12. Plot the graph and find out the wavelength of maximum absorbance.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 7. 9 . Select absorbance in the multi wavelength window then enter the wavelengths in a range of 200 to 400 at an interval of 5nm (increasing) for amino acids. 14. Cover the lid of the cuvette section in spectrophotometer. 13. Read and record the absorbance at each wavelength. 8. 10. Dispense the sample in cuvette and place it in sample cell.
SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . Other units such as Concentration Units (C. 'A.' or 'ABS'. or both.U. 580nm and 660nm.). all of which represent the same units. Turbidity has always been based on human observation and while this phenomenon is quantifiable by many different means. or haziness of the liquid. Absorbance units are commonly seen as 'A'. Turbidity is measured at 425nm. Take cuvette and hold it on its rough sides. 2.5 Objective: Determination of turbidity in the given sample by spectrophotometry. Clean the cuvette with distilled water and rinse with small amount of blank sample.D. Materials Required: Distilled Water sample MiliQ Water Drinking water sample Ground water sample Overnight grown bacterial culture UV-Vis Spectrophotometer Tissue paper Cuvettes Procedure: 1. 10 . Principle: Turbidity is an optical characteristic or property of a liquid. Turbidity is not colour related. The measuring path length or OPL is then set to work within the effective linear range of the instrument. much discussion still exits around the various techniques used to measure turbidities of fluids. with properly applied instruments.U.) can be measured at various pathlengths.5 grams per liter. LambertBeer hold true in most circumstances. Absorption based photometers are typically used for higher concentrations of process turbidity in excess of 0. also known as the spectrophotometer. colloidal material. Optical Density (O. Absorbance. is a device that measures the amount of light that can pass horizontally through the sample. which in general terms describes the clarity. The Colorimeter. but relates rather to the loss of transparency due to the effect of suspended particulate. being linear in most circumstances. In the case of solids concentration (turbidity). can then be easily correlated to any preferred unit of measure.
Observation Table: Sample Absorbance Turbidity Present/absent Result/ Interpretation: Interpret the results on the basis of absorbance values. 5. for different samples. Place it in the blank cell. 7. 6. 11 . Dispense the blank solution in cuvette wipe all the four walls with tissue paper. Dispense the sample in cuvette and place it in sample cell. Observation: Report the observation in following data table.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 3. Cover the lid of the cuvette chamber and read the absorbance at 425nm. 580nm and 660nm using spectrophotometer. Take another cuvette for sample. 4. Note down the O.D. 8. clean it and rinse with small aliquot of sample.
d = distance moved by the sample on the chromatogram. (Like dissolves like. Rf values of sample is defined as the relative distance traced by the sample with respect to the solvent front ( distanced moved by the solvent on the chromatographic paper). In paper chromatography the stationary phase is the filter paper and the mobile phase is the solvent. The filter paper holds the components until the solvent dissolves them and carries them up the filter paper. Since the amino acids are colorless in nature the samples will not be visible.6 Objective: Separation of Amino Acids by using Paper Chromatography and determination of their Rf values. D = distance moved by the solvent on the chromatogram. The solvent’s attraction to itself pulling it up is greater than the force of gravity pulling it down. To spot the samples after drying the chromatographic paper. Rf d D Where. ninhydrin solution is sprayed on to the chromatographic paper. All chromatographic techniques work on the same principle of affinity between the sample and either mobile phase or stationary phase. The solvent travels up the filter paper by capillary action. Since each component has its own solubility with the solvent and its own affinity to the solvent and filter paper. Depending on the solubility of sample in the solvent. Now the individual amino acids can be identified with reference to the marker samples or Rf values. The separation of components depends on their solubility with the solvent and their affinity to the solvent and filter paper. After some time the amino acid samples can be visualized due to the reaction between the amino acid and the ninhydrin solution resulting in the formation of a characteristic color.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . The solvent can only move the components if they are soluble in it and the more soluble a component is the more there is available to move up the filter paper. it will be dragged upwards along with the solvent and in this process the mixture of amino acid samples will get separated. Using this technique the individual amino acids can be separated from the mixture.) A component will travel up the filter paper at a rate that is determined by its affinity to the filter paper and solvent. 12 . Principle: Chromatography is used to separate mixtures of substances into the individual components. Solutes will dissolve into solvents that have similar properties. Polar solvents dissolve polar solutes and non-polar solvents dissolve non-polar solutes. Rf = Relative front. they can be separated in multiple ways by using mixtures of different solvents and different filter papers.
1) cm 2 dimensions. 10.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 Materials Required: Amino acids Ninhyrin reagent Paper chromatography sheets Acetone Solvent (Butanol.acetic acid-water 4:1:5(v\v)) Distilled water Hot air oven Glass chamber Marker pen Procedure: 1. Close the glass chamber with the lid and allow the solvent to climb up to the paper until it reaches to 2cm below the top edge. Mark a horizontal line at a distance of 2.0cm from the edge. Calculate the relative front for all the samples. S-2. Mark four spots on the horizontal line at equidistance and label them as S-1. Place the chromatographic paper in the middle of the chamber vertically leaning on to the wall of the chamber. Caution: Handle the chromatographic paper always using gloves only. Cut a piece of Chromatography paper into square shape strips about (7. 5.5cm. Load the amino acid samples on to the spots drop by drop using a micropipette and different tips for the respective amino acids and unknown samples. 7. Spot the colored samples on the chromatographic paper and measure the distance traveled by the solvent and the respective spots from the original marked horizontal line. 9. this may take up 20-30minutes time period 8. 11. Never touch the Chromatographic paper with bare hands. Take out the chromatographic paper from the oven and spray the ninhydrin solution on to the paper using a sprayer and allow it to dry. 13 . 2. 4. Pour the given solvent into the glass chamber to a depth of 1. 6.5 X 13. Take out the chromatographic paper out of the chamber and mark the solvent front with a pencil and then place it in the oven at 60 oC for 5 minutes. 3.
14 . No 1 2 Sample Distance moved (in cm) Rf Value Compare the Rf values and identify the components of the unknown sample mixture. Note: Clean all your glassware and the work bench after finishing your experiment. Result: Briefly write about the result and conclusion of the experiment performed.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 Observations: S.
0 it binds to negatively charged CM-Cellulose. At pH 7.5 ml of CM-Cellulose.+ M+ + A- The most popular method for the purification of proteins and other charged molecules is ion exchange chromatography. pipettes. 2. Ion exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. Procedure: Purification of Lysozyme using CM-cellulose: 1. resulting in the elution of lysozyme. Wash buffer with pH 9. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. Column. Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group: R-X-C+ + M+BR-X-M+ + C+ + B- Anion exchange chromatography retains anions using positively charged functional group: R-X+A. The ionic compound consisting of the cationic species M+ and the anionic species B.0 removes the protein that have pI equal to or less than 9. CM Cellulose. equilibration buffer. pI of lysozyme is 10.7 Objective: To separate the enzyme lysozyme by ion exchange chromatography.can be retained by the stationary phase . Fix the column to the stand.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography.+ M+BR-X+B. Wash the empty column with hot water (90°C). Cations compete with positively charged groups of lysozyme for binding sites on the column.5. elution buffer. test tubes etc.0. Materials Required: 1.5 and it carries a net positive charge at pH below 10. 15 . Remove the top cap of the column and pack the column with 2. wash buffer. Principle: Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. The lysozyme then is eluted by increasing concentrations of cations.
9. 12. 11. After an hour. 4. Take the crude enzyme preparation and adjust the pH to 7. 6. Remove the bottom cap and equilibrate the column with 50 ml of 1X equilibration buffer. Label this as eluate. Monitor OD at A280 and pool the fractions that show A280 0. Load rest of the supernatant to equilibrated CM-Cellulose column. Slowly pipette out or decant the supernatant without disturbing the column.5 and above. 3.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 3. 16 . Zero the spectrophotometer against phosphate buffer blank. Store at 4°C.77 x A260)] x dilution factor = ___________ mg/ml Result/ Interpretation: Report the concentration of protein in the given sample. Replace the top and bottom caps. Start collecting the eluate in test tubes as 5 ml fractions. for next use. Wash the column with approximately 30 to 40 ml of 1X wash buffer. Dilute the crude sample if necessary with PB 2. 4. Use the following formula to calculate concentration of protein in crude sample: Protein concentration = [(1. 10. Wash the column with 10 ml of 1M NaCl.0. Replace the top and bottom caps of the column and incubate for 1 hour at room temperature with intermittent mixing. Estimation of Protein Concentration: Crude sample: 1. 8. 5. Elute lysozyme from the column using 15 ml of 1X elution buffer. Measure the absorbance at A260 and A280.55 x A280) – (0. 7. allow the column material to settle.
Calcium sulphate 5. Material required: 1.0 mm for preparative TLC.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . 70% ethanol or spirit 3. 4. Gloves 9. Pencil 8. 2. 2.5 – 2. they give sharper separation and the amino acids or dipeptides can easily be collected from the plate. Thin layer chromatography is performed on a sheet of glass. Ninhydrin reagent 6. Prepare 50% silica gel in water. 17 . separation is achieved. a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. This layer of adsorbent is known as the stationary phase. Principle: Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures of biomolecules. which is coated with a thin layer of adsorbent material. TLC is replacing paper chromatography because the plates are easier to use than the paper. aluminium oxide. Chromatography chamber Procedure: 1. Dry the resultant plate and activate by heating in an oven for thirty minutes at 110 °C.25 mm for analytical purposes and around 0. Spread the gel as a thick slurry on glass slide. Solvent: n-Butanol: Glacial acetic acid: water :: 4: 1: 1 7. 3. Clean glass slides. After the sample has been applied on the plate. or cellulose (blotter paper). thick aluminum foil. Mark two spots on the line with a minimum distance of 1cm between them. Because different analytes ascend the TLC plate at different rates.1 – 0. or plastic to a thickness of around 0. or aluminum foil. Draw a line at one centimeter from one of the side of the glass plate.8 Objective: To separate and identify amino acids by thin layer chromatography. Silica gel 4. plastic. usually silica gel.
At the other spot apply standard known sample. 11. 18 . Allow the drops of samples to dry. which is dissolved and is carried up the plate by the solvent. Apply a small aliquot of sample solution to the plate on one of the spots. Place the plate in to a chromatography chamber containing solvent. 10. Once the solvent reaches to a sufficient distance (at least 3/4 th of height) remove the plate and mark the solvent front.) 9. Draw a centre for each spot and measure the distance between the point at which the sample was applied and the centre of the coloured spot (sample front). 14. (Keep ready the equilibrated chromatography chamber with the developing solvent for one hour. Allow the solvent to ascend through the silica gel.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 5. 6. 13. The solvent moves up the plate by capillary action and meets the sample mixture. Allow the plate to dry and then spray it with 2% ninhydrin solution. 12. Take out the plate from the oven. 8. Dry it again in an oven at 60°C for 5-10min. 7. Observation & Calculations: Note down the solvent front and the sample front and calculate the R f values of the standard known sample and that for the unknown sample. allow it to cool down and then encircle the coloured spots developed on the plate with the pencil. Result/ Interpretation: Match the values for identifying the unknown compound and report.
complexes get deposited in a very thin zone on the surface of the resolving gel. After migrating through the stacking gel of high porosity. Stain: Dissolve 0. Add 100 mL of acetic acid and. Destain: Add 500mL of HPLC. 2. The SDS-polypeptide complexes in the sample applied to the gel are swept along by a moving boundary created when an electric current is passed between the electrodes.9 Objective: To perform SDS. Acrylamide 3.polyacrylamide gel electrophoresis (SDS-PAGE) for separation of protein molecules. The final concentration of acetic acid is 5% (v/v). Storage solution: Add 25mL of acetic acid to 400mL of water. PAGE assembly Reagents Required: 1. Filter the solution to remove any insoluble material. The final concentration is 0. 19 .4g of Coomassie blue R350 in 200 mL of 40% (v/v) methanol in water with stirring as needed. adjust the total volume to 1000mL with water.1% (w/v) Coomassie blue R350. and 10% (v/v) acetic acid. On further electrophoresis. 20% (v/v) methanol. adjust the final volume to 500mL with water.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . Principle: SDS-PAGE: Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis (SDS-PAGE) is carried out in a discontinuous buffer system wherein the reservoir buffer is of a different pH and ionic strength from the buffer used to cast the gel. After mixing. after mixing. SDS 2. 3. Bis acrylamide 4. The final concentrations are 50% (v/v) methanol in water with 10% (v/v) acetic acid.grade methanol to 300 mL of water. Add 200mL of 20% (v/v) acetic acid in water. Materials Required: 1. polypeptides get resolved based on their size in the resolving gel.
Add 50 μl of APS solution to 5 ml of SDS separating gel mix and pour the gel solution between the plates till the level is 2 cm below the top edge of notched plate. 14. black: cathode. Silicon grease can be applied to spacer to make a water-tight seal. Fix the plates to PAGE apparatus. wash the top of the separating gel with distilled water and drain off the water completely. After the stacking gel has set.). This will take approximately 1 to 1 1/2 hours. Add 20 μl of APS solution to 2 ml of stacking gel mix and pour directly onto the polymerized separating gel. Note down the order of loading. 5. Fill the top reservoir with 1X reservoir buffer. Clamp the assembly of plates to fix it in a gel casting apparatus. Ensure the assembly is leak proof by filling water between the plates. 20 . Fill the bottom reservoir with 1X reservoir buffer and carefully fix the plate to the apparatus without trapping any air bubbles between the buffer and the bottom of the gel. 40 μl of protein sample in well 2 and 5 μl of protein sample in well 4. 2. Wash the wells immediately with distilled water to remove non-polymerized acrylamide. Set voltage at 100 V and switch on the power supply. Add 25 μl of sample loading buffer to protein sample. not at the notch. 10. 9. Add 25 μl of sample loading buffer to 25 μl of protein marker. 11. When the dye front comes to 0. The stacking gel will set in approximately 10 min. Assemble the plates for casting gel as shown below. Insert the comb into the gel solution carefully without trapping any bubbles. 8. 4.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 Day 1: SDS-PAGE 1. turn off the power. Remove the gel plates and gently pry the plates apart using a spatula or similar tool. Connect the cords to the power supply according to the convention red: anode. After the gel is set (approximately 20-30 min. Load 30 μl protein markers in well 1. 3.5 cm above the bottom of the gel. carefully remove the comb and the bottom spacer. 13. Add 200 to 250 μl of water to make the surface even. 15. 6. 12. about 1cm above the separating gel. 7. Place it in a boiling water bath for 5 minutes.
18. Stain the gel at room temperature for 1 to 2 hr with gentle agitation.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 16. Note: For uniform staining and washing. The destain solution should be changed several times. 21 . 21. 17. The gel should return to its original dimensions during this process. It might be convenient to carefully transfer the gel to a heat-sealable bag for longer-term storage. Note: Cover the tray and leave it overnight at room temperature. Remove the staining solution and cover the gel with the destain solution and allow the gel to destain with gentle agitation for 3-4hrs. 20. Transfer the gel to a tray containing the Coomassie stain. Store the gel in the storage solution as needed. place the tray on a rocker or shake intermittently every 10 to 15 minutes. removing it at each change by aspiration. 19. Continue the destaining until the protein bands are seen without background staining of the gel. Equilibrate the gel in the storage solution for at least 1 hr.
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