This action might not be possible to undo. Are you sure you want to continue?
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.
Take cuvette and hold it on rough sides. Considering intensity of transmitted light as I and that of incident light as I0. A = log10 I0 . 6. it can be said that optical density is proportional to the sample concentration if the path-length is constant. 3.D. Stating the Beer-Lambert law differently. 150 μl and respectively increasing amount upto 800 μl in other tubes (tube no. When high sample concentrations are being measured. Procedure: 1. 2. Take 5ml of Q3 water in tube no. i. Beer’s Law: The amount of light absorbed by a material is proportional to the number of absorbing molecules. Working DNA solution (500μg/ml): Dilute 10ml of stock solution to 100ml.e. Mix the contents. the molecules may dimerize or aggregate which causes positive or negative deviation. 5.log10 I Absorbance shares linear relationship with sample concentration. Materials required: 1. 3 .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. 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. 4. 16. 1. 2 to 15). Take 100 μl of working DNA in tube no. Stock DNA solution: Dissolve 50mg of DNA in 10ml of Q3 water to get a final concentration of 5mg/ml. the concentration of the absorbing solution. Make up the volume to 5ml using Q3 water. and the reverse I/I0 is known as Transmittance. Place 16 clean dry test tubes in test tube stand and label them 1 – 16. the quantity I0/I is known as O. 2. 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).
4 .9 10 2 150 4. clean it and rinse with small aliquote of sample.7 30 6 350 4. D.8 20 4 250 4. (260nm) 1 100 4.35 65 13 700 4.55 45 9 500 4.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 7.D. Working DNA solution (μl) Distilled water (ml) Final conc.3 70 14 750 4. 9. Dispense the blank solution in cuvette wipe all the four walls with tissue paper. Protocol: Name of the student: Test Tube No.7 5 25 5 300 4. 8. Place it in the blank cell.45 55 11 600 4.5 50 10 550 4.4 60 12 650 4.85 15 3 200 4.00 Result/Interpretation: From the graph verify the Beer Lambert Law. Cover the lid of the spectrophotometer and read the absorbance at 260nm using spectrophotometer.2 80 16 Blank 5. Clean the cuvette with distilled water and rinse with small amount of Q3 water (Blank). Dispense the sample in cuvette and place it in sample cell. with respect to the concentrations and plot the graph with the readings against the concentration.25 75 15 800 4. 11. Take another cuvette for sample. Note down the O. 10.65 35 7 400 4. μg/ml O. 13. 12.0 0.6 40 8 450 4.
usually abbreviated as EtBr. 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. or RNA molecules by size.g. The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide. e. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light.3 Objective: To perform Submerged Agarose Gel Electrophoresis for Separation and Detection of Nucleic Acids. The technique is used for estimation of the size of DNA molecules following restriction enzyme digestion. e.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . Bromophenol blue 25mg 5 . or of RNA prior to Northern transfer.g. analysis of PCR products. any band containing more than ~20ng DNA becomes distinctly visible. Shorter molecules move faster and migrate farther than longer ones. Principle: Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA. in restriction mapping of cloned DNA. Separation of restricted genomic DNA prior to Southern transfer. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). in molecular genetic diagnosis or genetic fingerprinting.
When the gel has cooled down and become solid. 6 . 7. 1. 4.0): Tris Base Glacial Acetic acid EDTA (0. The holes that remain in the gel are the wells or slots. Let the solution cool down to about 50 °C at room temperature. Insert the comb at one side of the gel.25M) Volume make up to 242 g 57. Mount the gel. or water bath. Wear gloves from here on. Mix it with 150 ml 1x TAE buffer and then heat in a microwave oven or on hot plate until completely melted. 5. 2.0x.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 2. Sucrose 4. carefully remove the comb. Be very careful when handling the concentrated stock. together with the rack.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. ethidium bromide is a mutagen. Xylene cyanol 3.5g of Agarose powder in a 250ml conical flask. pH 8. Stir the solution to disperse the ethidium bromide. Then pour it into the gel rack. Stir or swirl the solution while cooling. 6. 3.0 Composition of stock TAE 50x (pH 8.5 μg/ml. about 5-10 mm from the end of the gel. into electrophoretic tank containing 1x TAE. Add 5 µl ethidium bromide stock (10 mg/ml) per 100 ml gel solution for to the gel at a final concentration of 0. Distilled water 25mg 4g 10ml Buffer solution: TAE 1.
Load the DNA molecular weight marker (DNA Ladder) mixed with loading dye beside the sample well. turn off the electric current and remove the electrodes and cover from the gel tank. DNA will migrate towards the positive electrode (anode) which is usually colored red. The gel must be completely covered with TAE. 11. 16. 14. Mix the sample of DNA with 0.2 volume of the 6x gel-loading buffer (eg. Place the cover over the tank and connect the electrode wires at the respective positions in the electrophoretic tank and to the power pack. Examine the gel on UV transilluminator and document with the gel documentation system. 13. 9. with the slots at the end electrode that will have the negative current. Observation: Report the observation from the image obtained after documentation. Load the DNA samples mixed with loading buffer slowly into the sample wells using micropipette. Mix 5μl DNA sample with 1μl gel loading buffer). 15. 7 .SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 8. Result/ Interpretation: Report the result in terms of separation and detection analysis. Apply 50V current and set the timer for 2h. 10. When the DNA samples or dyes have migrated a sufficient distance through the gel. Confirm the flow of current by observing bubbles coming off the electrodes. 12.
Principle: When sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule. Switch on the spectrophotometer and give a warm up time of 30min.4 Objective: To Determination of absorption maxima (λmax) of the given sample by UV-Visible spectrophotometer. Take cuvette and hold it on rough sides. 8 . Place it in the blank cell.1mg/ml) Bovine serum albumin (0.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . Absorbance usually ranges from 0 (no absorption) to 2 (99% absorption).1mg/ml) UV-Vis Spectrophotometer Tissue paper Cuvettes MiliQ Water Procedure: 1.1mg/ml) NADH (0. 6. 2. Clean the cuvette with distilled water and rinse with small amount of blank sample (MiliQ water). Dispense the blank solution in cuvette wipe all the four walls with tissue paper. This pattern is known as absorption spectrum. some of the light energy will be absorbed as the electron is promoted to a higher energy orbital. The pattern of energy absorption by a substance when light of varying wavelength passes through it is uniquely characteristic of substance.1mg/ml) Bromophenol blue solution (0. The wavelength at which the compound absorbs most is known as its absorption maxima. Prepare the amino acid solutions in MiliQ water. 4. The resulting spectrum is presented as a graph of absorbance (A) versus wavelength. and is precisely defined in context with spectrometer operation. Phenylalanine) solutions in MiliQ water (0. 5. 3. Materials Required: Amino acid (Lysine.
11. 12. 14. Observation: Report the observation in following data table and plot the graph using the absorbance values against the wavelength. 9. 13. Read and record the absorbance at each wavelength. Dispense the sample in cuvette and place it in sample cell. 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. Take another cuvette for sample. Switch on the UV and Vis lights and select the multi wavelength option from the main menu in spectrophotometer. For Bromophenol blue solution read the absorbance in the range of 300 – 700nm.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 7. clean it and rinse with small aliquote of sample. Data Table: Wavelength () Absorbance Wavelength () Absorbance Result/Interpretation: Determine the peak on the graph and report the absorption maxima for the given amino acid. 10. Cover the lid of the cuvette section in spectrophotometer. 8. Plot the graph and find out the wavelength of maximum absorbance.
The Colorimeter.U. Absorbance. Principle: Turbidity is an optical characteristic or property of a liquid.U.' or 'ABS'.). but relates rather to the loss of transparency due to the effect of suspended particulate. In the case of solids concentration (turbidity). Absorption based photometers are typically used for higher concentrations of process turbidity in excess of 0.5 grams per liter. Turbidity has always been based on human observation and while this phenomenon is quantifiable by many different means. or haziness of the liquid. 10 . Other units such as Concentration Units (C. is a device that measures the amount of light that can pass horizontally through the sample. Absorbance units are commonly seen as 'A'.D. can then be easily correlated to any preferred unit of measure. 580nm and 660nm. Turbidity is not colour related. Clean the cuvette with distilled water and rinse with small amount of blank sample. or both. Take cuvette and hold it on its rough sides. Optical Density (O. being linear in most circumstances. all of which represent the same units. 2. colloidal material. 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. LambertBeer hold true in most circumstances. 'A. much discussion still exits around the various techniques used to measure turbidities of fluids. with properly applied instruments.5 Objective: Determination of turbidity in the given sample by spectrophotometry. which in general terms describes the clarity. Turbidity is measured at 425nm. also known as the spectrophotometer.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT .) can be measured at various pathlengths. The measuring path length or OPL is then set to work within the effective linear range of the instrument.
Cover the lid of the cuvette chamber and read the absorbance at 425nm. Place it in the blank cell. 6. Note down the O. 580nm and 660nm using spectrophotometer. Observation: Report the observation in following data table. 7. Dispense the sample in cuvette and place it in sample cell.D. Take another cuvette for sample. Dispense the blank solution in cuvette wipe all the four walls with tissue paper. 8. Observation Table: Sample Absorbance Turbidity Present/absent Result/ Interpretation: Interpret the results on the basis of absorbance values. 11 . 4. for different samples. 5. clean it and rinse with small aliquot of sample.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 3.
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. Using this technique the individual amino acids can be separated from the mixture. Principle: Chromatography is used to separate mixtures of substances into the individual components. it will be dragged upwards along with the solvent and in this process the mixture of amino acid samples will get separated. The solvent travels up the filter paper by capillary action. D = distance moved by the solvent on the chromatogram.) A component will travel up the filter paper at a rate that is determined by its affinity to the filter paper and solvent. 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. (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). All chromatographic techniques work on the same principle of affinity between the sample and either mobile phase or stationary phase. Solutes will dissolve into solvents that have similar properties. The separation of components depends on their solubility with the solvent and their affinity to the solvent and filter paper. Rf d D Where. d = distance moved by the sample on the chromatogram. Since the amino acids are colorless in nature the samples will not be visible. they can be separated in multiple ways by using mixtures of different solvents and different filter papers. Polar solvents dissolve polar solutes and non-polar solvents dissolve non-polar solutes.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . The filter paper holds the components until the solvent dissolves them and carries them up the filter paper. 12 . To spot the samples after drying the chromatographic paper. 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. ninhydrin solution is sprayed on to the chromatographic paper.6 Objective: Separation of Amino Acids by using Paper Chromatography and determination of their Rf values. The solvent’s attraction to itself pulling it up is greater than the force of gravity pulling it down. Rf = Relative front. In paper chromatography the stationary phase is the filter paper and the mobile phase is the solvent.
S-2.0cm from the edge. Caution: Handle the chromatographic paper always using gloves only.5cm. Place the chromatographic paper in the middle of the chamber vertically leaning on to the wall of the chamber. this may take up 20-30minutes time period 8. 13 . 9. 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. 10. 4. 11. 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. 6.acetic acid-water 4:1:5(v\v)) Distilled water Hot air oven Glass chamber Marker pen Procedure: 1.5 X 13. 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.1) cm 2 dimensions.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. Mark four spots on the horizontal line at equidistance and label them as S-1. Cut a piece of Chromatography paper into square shape strips about (7. Calculate the relative front for all the samples. Mark a horizontal line at a distance of 2. 7. 3. 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. Never touch the Chromatographic paper with bare hands. 5. 2. Pour the given solvent into the glass chamber to a depth of 1. 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.
Result: Briefly write about the result and conclusion of the experiment performed. Note: Clean all your glassware and the work bench after finishing your experiment. No 1 2 Sample Distance moved (in cm) Rf Value Compare the Rf values and identify the components of the unknown sample mixture. 14 .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. Cations compete with positively charged groups of lysozyme for binding sites on the column. The ionic compound consisting of the cationic species M+ and the anionic species B. Wash buffer with pH 9. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography.5 ml of CM-Cellulose. Ion exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. resulting in the elution of lysozyme. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. 2. Remove the top cap of the column and pack the column with 2.+ M+BR-X+B. elution buffer.5 and it carries a net positive charge at pH below 10. Principle: Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their 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. pI of lysozyme is 10. Fix the column to the stand.5.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . pipettes. wash buffer. test tubes etc. 15 . Procedure: Purification of Lysozyme using CM-cellulose: 1.0 removes the protein that have pI equal to or less than 9. Materials Required: 1. At pH 7.7 Objective: To separate the enzyme lysozyme by ion exchange chromatography.0.can be retained by the stationary phase . The lysozyme then is eluted by increasing concentrations of cations. Wash the empty column with hot water (90°C). CM Cellulose.+ M+ + A- The most popular method for the purification of proteins and other charged molecules is ion exchange chromatography. equilibration buffer.
16 . 7. Replace the top and bottom caps of the column and incubate for 1 hour at room temperature with intermittent mixing. Start collecting the eluate in test tubes as 5 ml fractions. Remove the bottom cap and equilibrate the column with 50 ml of 1X equilibration buffer. 9.5 and above. Elute lysozyme from the column using 15 ml of 1X elution buffer. Wash the column with approximately 30 to 40 ml of 1X wash buffer. Slowly pipette out or decant the supernatant without disturbing the column.0. Label this as eluate. 6. 10. 3.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 3. 4. Zero the spectrophotometer against phosphate buffer blank. Dilute the crude sample if necessary with PB 2.77 x A260)] x dilution factor = ___________ mg/ml Result/ Interpretation: Report the concentration of protein in the given sample. After an hour. Load rest of the supernatant to equilibrated CM-Cellulose column. 12. allow the column material to settle. Take the crude enzyme preparation and adjust the pH to 7. 4. for next use. Replace the top and bottom caps. 11. Wash the column with 10 ml of 1M NaCl. Estimation of Protein Concentration: Crude sample: 1. Monitor OD at A280 and pool the fractions that show A280 0. Use the following formula to calculate concentration of protein in crude sample: Protein concentration = [(1. Store at 4°C.55 x A280) – (0. 8. 5. Measure the absorbance at A260 and A280.
0 mm for preparative TLC. 4. Ninhydrin reagent 6. a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Draw a line at one centimeter from one of the side of the glass plate.25 mm for analytical purposes and around 0.8 Objective: To separate and identify amino acids by thin layer chromatography. After the sample has been applied on the plate. plastic. Dry the resultant plate and activate by heating in an oven for thirty minutes at 110 °C. This layer of adsorbent is known as the stationary phase. they give sharper separation and the amino acids or dipeptides can easily be collected from the plate. Gloves 9. Pencil 8.1 – 0. thick aluminum foil. separation is achieved. Thin layer chromatography is performed on a sheet of glass. 17 . Clean glass slides. Because different analytes ascend the TLC plate at different rates. Solvent: n-Butanol: Glacial acetic acid: water :: 4: 1: 1 7. or cellulose (blotter paper). 3. Silica gel 4. Spread the gel as a thick slurry on glass slide. TLC is replacing paper chromatography because the plates are easier to use than the paper. Chromatography chamber Procedure: 1. Principle: Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures of biomolecules. aluminium oxide. 70% ethanol or spirit 3.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT .5 – 2. usually silica gel. Mark two spots on the line with a minimum distance of 1cm between them. Material required: 1. or plastic to a thickness of around 0. or aluminum foil. which is coated with a thin layer of adsorbent material. 2. 2. Prepare 50% silica gel in water. Calcium sulphate 5.
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. Take out the plate from the oven. Apply a small aliquot of sample solution to the plate on one of the spots. 12.) 9. (Keep ready the equilibrated chromatography chamber with the developing solvent for one hour. 18 . 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). Place the plate in to a chromatography chamber containing solvent. The solvent moves up the plate by capillary action and meets the sample mixture. At the other spot apply standard known sample. 7. 10. 6. 13. Allow the drops of samples to dry. 8. 14. 11.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 5. Allow the plate to dry and then spray it with 2% ninhydrin solution. Result/ Interpretation: Match the values for identifying the unknown compound and report. which is dissolved and is carried up the plate by the solvent. allow it to cool down and then encircle the coloured spots developed on the plate with the pencil. Once the solvent reaches to a sufficient distance (at least 3/4 th of height) remove the plate and mark the solvent front. Allow the solvent to ascend through the silica gel. Dry it again in an oven at 60°C for 5-10min.
19 . Add 200mL of 20% (v/v) acetic acid in water. polypeptides get resolved based on their size in the resolving gel. Acrylamide 3. Materials Required: 1.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 EXPERIMENT . 3. After mixing. adjust the final volume to 500mL with water. On further electrophoresis. and 10% (v/v) acetic acid. 2. 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. Filter the solution to remove any insoluble material.grade methanol to 300 mL of water. complexes get deposited in a very thin zone on the surface of the resolving gel. 20% (v/v) methanol. adjust the total volume to 1000mL with water. Add 100 mL of acetic acid and. The final concentration of acetic acid is 5% (v/v).1% (w/v) Coomassie blue R350. The final concentrations are 50% (v/v) methanol in water with 10% (v/v) acetic acid. Destain: Add 500mL of HPLC.polyacrylamide gel electrophoresis (SDS-PAGE) for separation of protein molecules. SDS 2.9 Objective: To perform SDS. after mixing. Stain: Dissolve 0. PAGE assembly Reagents Required: 1.4g of Coomassie blue R350 in 200 mL of 40% (v/v) methanol in water with stirring as needed. After migrating through the stacking gel of high porosity. Bis acrylamide 4. Storage solution: Add 25mL of acetic acid to 400mL of water. 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. The final concentration is 0.
Insert the comb into the gel solution carefully without trapping any bubbles. 10. black: cathode. 8. Assemble the plates for casting gel as shown below. Ensure the assembly is leak proof by filling water between the plates. about 1cm above the separating gel. wash the top of the separating gel with distilled water and drain off the water completely. Set voltage at 100 V and switch on the power supply. 2. Add 200 to 250 μl of water to make the surface even. Add 20 μl of APS solution to 2 ml of stacking gel mix and pour directly onto the polymerized separating gel.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 Day 1: SDS-PAGE 1.). 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. not at the notch. After the stacking gel has set. 40 μl of protein sample in well 2 and 5 μl of protein sample in well 4.5 cm above the bottom of the 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. 15. Silicon grease can be applied to spacer to make a water-tight seal. 7. Connect the cords to the power supply according to the convention red: anode. 3. Add 25 μl of sample loading buffer to 25 μl of protein marker. 6. 5. Wash the wells immediately with distilled water to remove non-polymerized acrylamide. 14. Place it in a boiling water bath for 5 minutes. Note down the order of loading. Fill the top reservoir with 1X reservoir buffer. When the dye front comes to 0. 13. Add 25 μl of sample loading buffer to protein sample. Fix the plates to PAGE apparatus. 9. turn off the power. Remove the gel plates and gently pry the plates apart using a spatula or similar tool. The stacking gel will set in approximately 10 min. 12. After the gel is set (approximately 20-30 min. This will take approximately 1 to 1 1/2 hours. carefully remove the comb and the bottom spacer. 11. Load 30 μl protein markers in well 1. Clamp the assembly of plates to fix it in a gel casting apparatus. 20 . 4.
Remove the staining solution and cover the gel with the destain solution and allow the gel to destain with gentle agitation for 3-4hrs. place the tray on a rocker or shake intermittently every 10 to 15 minutes. Equilibrate the gel in the storage solution for at least 1 hr. 19. Note: For uniform staining and washing. Stain the gel at room temperature for 1 to 2 hr with gentle agitation. 21. The destain solution should be changed several times. 17. removing it at each change by aspiration. Transfer the gel to a tray containing the Coomassie stain. 20. It might be convenient to carefully transfer the gel to a heat-sealable bag for longer-term storage.SPSU text] [Type UDAIPUR Department of Biotechnology LAB MANUAL-Analytical Techniques in Biotechnology BT-202 16. Store the gel in the storage solution as needed. The gel should return to its original dimensions during this process. 18. Note: Cover the tray and leave it overnight at room temperature. 21 . Continue the destaining until the protein bands are seen without background staining of the gel.
This action might not be possible to undo. Are you sure you want to continue?