Lab manual

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Lab manual

  1. 1. NAME OF THE EXPERIMENTS1. BUFFER PREPARATION2. ESTIMATION OF PROTEIN BY LOWRY’S METHOD3. ABSORPTION MAXIMA4. PAPER CHROMATOGRAPHY5. PH TITRATION6. PROTEIN PURIFICATION BY AMMONIUM SULFATE PRECIPITATION7. ION EXCHANGE CHROMATOGRAPHY8. SDS – PAGE9. MOLECULAR MODELLING 2
  2. 2. BUFFER PREPARATIONAIM: To prepare phosphate buffer (pH=7.5) and acetate buffer (pH=5).PRINCIPLE: A buffer is a solution which reacts which resists small change in hydrogenconcentration of acid or base. Buffer solutions are the mixture of weak acids and their saltsor conjugate base. The conjugate base provided by salt dissociation is actually involved inthe following buffering action. The capacity of a buffer to resist the change in pH depends on the actualconcentration of a buffer. Salt to acid concentration ration when it’s utility say 1:1 the bufferhas maximum efficiency.The buffering range of any given buffer is in PH units on either side of the pKa of the bufferacid.REAGENTS REQUIRED:Sodium hydrogen phosphate (NaH2PO4) – acid – 0.1MDisodium hydrogen phosphate (Na2HPO4) – Base – 0.1MSodium acetate (CH3COONa) – acid – 0.1MAcetic acid (CH3COOH) – Acid – 0.1M 3
  3. 3. PROCEDURE:PHOSPHATE BUFFER • 0.1M of NaH2PO4 and Na2HPO4 solutions were prepared separately. • To 50mL of 0.1M Na2HPO4, add 0.1M NaH2PO4 and check pH using pH meter. • The pH was brought to 7.5 by using 0.1M NaH2PO4 solution and the entire volume was made upto 500mL using distilled water.ACETATE BUFFER: • 0.1M CH3COONa and CH3COOH solution is prepared separately. • To 50 mL of 0.1M CH3COONa 300ml of distilled water is added and its pH checked by a pH meter. • The pH is brought to 5 by using 0.1M CH3COOH solution. The final volume is made 500mL with distilled water.RESULT: 500mL of 0.1M phosphate buffer at pH 7.5 and 0.1M acetate buffer of 500mL volumeat pH 5 were prepared separately. 4
  4. 4. ESTIMATION OF PROTEIN BY LOWRY’S METHODAIM: To estimate the amount of protein in a solution.PRINCIPLE: Sodium tungstate, Molybdate and phosphate present in folin ciocateau reagentreacts with phenolic group of tyrosine residues in protein to produce a blue purple colour.This has an absorption maximum at 640nm. This method is sensitive upto approximately 10g/mL of protein concentration. This assay is also sensitive to pH. (Working range pH 10 –10.5) and length of incubation time.REAGENTS:Solution 1: 2% of Sodium carbonate (w/v) keep in 0.1 NaOHSolution 2: 4% NaK tartarate (w/v) in distilled waterSolution3: 2% CuSo4 in distilled waterFolin ciocateau 50ml in 50mL of waterBovine serum albumin (BSA) standard: 400µg/mLPREPARATION OF ALKALINE REAGENT (ALKALINE COPPER) The solution 1, 2, 3 are mixed in the ratio of 100:1:1 to prepare alkaline reagent.FOLIN CIOCALTEAU REAGENT: Commercially available reagent is diluted to 50% by distilled water and used. 5
  5. 5. BSA STANDARD SOLUTION:4mg of BSA is dissolved in 1ml of distilled water to get a concentration of 400µg/mL.PROCEDURE: • To a series of labelled (Duplicated) testes s1 – s5. BSA solution is added in the range 0.1 mL– 0.5mL. • The tubes are made up to 0.5mL by adding distilled water. • The tube with 0.5mL of distilled water serves as blank. • The 5mL of alkaline copper reagent is added to all the tubes including test which contains 0.1mL of test sample, 0.4ml of distilled water. • All the tubes are incubated at room temperature for 20 minutes. • After the incubation 0.5mL of Folin’s reagent is added to each tube and re-incubated for 15minutes. • Absorbance is noted by setting the spectrophotometer to 0% absorbance for blank. • A standard graph is plotted by tracing the O.D values on Y - axis and increasing by X - axis. • The test samples the OD is spotted on the standard and perpendicular is drawn towards concentration. • The concentration of protein in this sample can be calculated using the formula. Amount of protein in test tube= OD of test x conc. of standard x Dilution factor OD of standardRESULT: Amount of protein present in the given solution according to the graph is ………………….The amount of protein calculated by the formula is …………………. 6
  6. 6. ABSORPTION MAXIMAAIM: To determine the wavelength of maximum absorption for the sample solution riboflavin.PRINCIPLE: Every particle in the universe has the property of absorption of light. If differs fromatom to atom of different elements where they can absorb the light rays maximum. Suchthat all of its atom are there in excited state. The wavelength at which, a solution ofsubstance absorbs maximum amount of light is called its absorption maximum.MATERIALS REQUIRED: Spectrophotometry Riboflavin – 5mg/mLPROCEDURE: • The riboflavin solution is taken in a test tube 1mL and to it 14 mL of water is added to make up to volume of 15mL. • The absorbance of the sample at different wavelength 260 – 650 nm were taken on UV visible spectrophotometry.RESULT: Maximum absorption of riboflavin was founded at …………….. 7
  7. 7. PAPER CHROMATOGRAPHYAIM: To separate a mixture of amino acids by using a paper chromatography.PRINCIPLE: Cellulose in the form of paper sheets makes an ideal support medium where water isabsorbed between the cellulose fibres and form the stationary phase. The mobile phase issecondary butanol, acetic acid and water in the ratio of 4:1:5. The different amino acid in the mixture are separated based on the difference intheir relative solubility between the stationary phase and the mobile phase. If is the capillaryaction which allows the movement of the solvent and sample in the paper.REQUIREMENTS:Whatmann no. 1 filter paperSolvent system (Butanol: Acetic acid: water in 4:1:5 ratioNinhydrin (0.25g in acetone (0.2%)Distilled waterAmino acid sampleSpot lamp 8
  8. 8. PROCEDURE: • The machine direction of the paper is observed and cut into many strips in that direction. • The sample containing the mixture of amino acids are spotted with capillary tube (for maximum precision) on a paper at a distance of about 1 inch away from the edge to be dipped in the solvent system. • The spot with amino acid sample is concentrated by repeated spotting and drying. Then the solvent system is kept in chromatographic chamber which is already saturated with solvent system vapours. • The chromatographic paper spotted with amino acid samples is dipped in the solvent system upto 1 cm from the bottom of chamber. • The solvent is made to run under capillary action such that it travels 2/3 portion of the paper. The paper is then removed and exposed to NH3 vapours for neutralization and allowed to dry. • After drying 0.2% Ninhydrin is sprayed on the dried chromatogram and purple coloured spots are observed on the chromatogram. • The distance travelled by the solvent system and the distance travelled by the respective amino acids from the point of spotting is scaled and Rf is calculated. Rf value = Distance moved by the solute D Distance moved by the solventRESULT: The amino acids were separated in different spaces in chromatogram. 9
  9. 9. PH TITRATIONAIM: To estimate the change in PH while titrating weak acid against strong base.PRINCIPLE: pH signifies potential of hydrogen atoms. It’s the negative log of hydrogen ionsconcentration. Acetic acid is a weak acid, it doesn’t undergo protonation easily as ‘ OH’never gets completely ionised, along with positive ionic effect of adjacent (CH3) groupattached to carbon atom. The H+ of acid gets neutralized with –OH of strong base. Theamount of NaOH needed to bring about neutralization gives PH of that particular acid.CH3COOH  CH3COO- +H+ NaOH  Na+ +OH- CH3COOH+NaOHCH3COONa+ H2OMATERIALS REQUIRED: pH meter 0.1M NaOH solution 0.1M acetic acid solution Beaker Pipette 10
  10. 10. PROCEDURE:  Take 10mL of 0.1N acetic acid solution in a clean beaker.  The pH meter is standardised using standard pH or buffer of known pH.  The pH of acetic acid is noted.  Then add 1mL of NaOH to 0.1N acetic acid in beaker with constant stirring or mixing drop by drop of NaOH to CH3COOH. The pH is noted.  Like above mentioned add 1mL of NaOH at regular interval and pH noted with corresponding addition of NaOH upto it reaches alkalinity.RESULT: pH titration was performed. The pKa of the acetic acid is ………………… 11
  11. 11. PROTEIN PURIFICATION BY AMMONIUM SULPHATE PRECIPITATIONAIM:To purify proteins at different concentration of ammonium sulphate and estimate it byLowry’s method.PRINCIPLE: Basically the protein precipitation depends upon salting out of ammonium sulphateon addition of salt of ammonium sulphate on addition of salt to protein the shell ofhydration of proteins is removed. This causes the precipitated amount of protein areestimated by Lowry’s method.REQUIREMENTS:Ammonium sulphate 10g, 20g, 30g weight separately.Protein standard 10mg, 20mg of protein per mL.Copper sulphate 10%Sodium potassium tartrate 2%Na2CO3 2% of Na2CO3 in 0.1N NaOHFolin reagentSTANDARD SOLUTION 1mg of BSA in distilled water. 12
  12. 12. Coomessive dye reagent CBB – G250 100mg Methanol 50mL Orthophosphoric acid 100mL Distilled water make upto 1000mLPROCEDURE: Three cleaned beakers containing 10%, 20%, 30% ammonium sulphate are taken.They are added in succession 100ml BSA solution taken in different contents and constantstirring is done while adding the salt solution. The solutions are then allowed to stand for1 hour at room temperature. An adequate of 1.5ml from each beaker is taken and centrifuged at 1000rpm for 510minutes. The supernatant is discarded. The pallet is suspended in 10mL of distilled water. The protein content of pallet isquantitatively estimated by using barfoed’s method. The values are plotted on a standardgraph.RESULT: The amount of protein in the given sample by an ammonium sulphate precipitationis……………. 13
  13. 13. ION EXCHANGE CHROMATOGRAPHYAIM: To separate amino acids by ion – exchange column chromatography using anionexchanger.PRINCIPLE: Ion exchange chromatography can be used for separation of substances which passesa net electrical charge. Anion exchanger reversibly bind negatively charged compoundsthrough electrostatic forces whereas positively charged ones interact with cation exchanger.Different compounds are held by ion exchangers with varying strengths depending uponcharge. Amino acids are amphoteric substances and have a net charge of zero at isoelectricpH. At pH below isoelectric point the amino acids are positively charged while ate higher pHthey exist as anions. Hence the pH of system will be high, above isoelectric point whereamino acids exists as anions and can be separated on an anion exchanger.REQUIREMENTS:Chromatography columnAmberlite IR-200 polystyreneNinhydrin reagentGlass woolAmino acid mix 2mg of each amino acid per ml of 0.1N HCL at pH 1.0 14
  14. 14. PROCEDURE:PREPARATION OF ION EXCHANGER – AMBERLITE- IR 200 Suspend 10g of amberlite IR 200 into sufficient volume of phosphate buffer for15minutes to ensure that the resin is saturated with chloride.EQUILIBRATION OF RESINS: • Suspend the resin in phosphate buffer of pH 7.4 and allow it to stand for 1hour. • Mount the column upright and pour suspension with the help of glass rod while tapping column gently. • Allow the suspension to settle down. Open the outlet and pass two to three volumes of phosphate buffer (0.1 M, pH 7.4). This will fully equilibrate the resin to pH 7.4. When only a thin layer of buffer remains at the top of the resins, stop the flow by closing column outlet.SAMPLE LOADING: Open the outlet and let the buffer at the top to drain the column surface. Close thestopcock.DEVELOPMENT OF COLUMN: All the amino acids would be in anionic form at pH 7.4, so would be bound to anionexchanger. Gradient elution using increasing pH or 0.5M NaOH solution in buffer solutionused to facilitate sequential elution of the bound amino acids.RESULT: The given amino acid mixture were separated by spraying Ninhydrin on the aminoacid spots placed on the whatmann No 1 filter paper. Amino acid concentration can bedetermined by observing the intensity of purple colour. 15
  15. 15. SDS – PAGEAIM: To perform SDS – PAGE.PRINCIPLE: Sodium dodecyl sulphate (SDS) is an anionic detergent which binds to protein andconfers negative charge uniformly along its entire length.One molecule of SDS binds to two amino acid residues of proteins. As proteins gain uniformnegative charge, so the rate of migration towards anode is totally determined by themolecular weight.MATERIALS REQUIRED:30% ACRYLAMIDE STOCK SOLUTION acrylamide 30g Bis acrylamide 0.8g Distilled water 100mLRESOLVING GEL BUFFER (pH 8.8) Tris buffer base 22.7g of 1.875M Distilled water 70mL then adjust pH to 8.8 Make upto 100ml using Distilled water.STACKING GEL BUFFER (pH 6.8) Tris buffer HCL 7.2g of 0.6M Distilled water 70mL Adjust pH 6.8 and make the volume upto100mLPOLYMERIZATION AGENTS: 5% APS Dissolve 5g ammonium per sulphate in 100mL of waterTEMED N,N,N’,N’ tetra methyl ethylene diamine used directly from the refrigerator.ELECTRODE BUFFER (pH 8.2 – 8.4) Tris base 12g of 0.005M Glycine 28.8g of 0.192M 16
  16. 16. Distilled water 200mLSAMPLE BUFFER (5x) Stacking gel buffer 5mL SDS 0.5g Mercaptoethanol 0.25mL Bromophenol blue 1mL (0.5% w/v) Distilled water 10mL store in frozen aliquots.10% SDS SOLUTION SDS 10g Distilled water 100mLSTANDARD PROTEIN 1g of BSA in 1mL of distilled water.STAINING SOLUTION (R250) coomassive brilliant blue dye 0.1g Methanol 40mL Acetic acid 10mL Distilled water 50mL DESTAININGSOLUTION Methanol 40mL Acetic acid 10mL Distilled water 50mLPROCEDURE  ASSEMBLING OF GLASS PLATES o Thoroughly clean the glass plates and the spacers and assemble them properly. o Hold the assembly together and damp them in upright position. o Apply 2% agar or 17
  17. 17. petroleum gel around the edges of the spacers to hold them in place and seal the space between glass plates.  PREPARATION OF RESOLVING GEL (30mL) o 13.3mL of Acrylamide stock is added to 8.0mL of resolving gel buffer (pH 8.8) and 18.1mL of distilled water. o To this mixture, add 5% APS 0.2mL 10%SDS 0.4mL TEMED 20µL  STACKING GEL PREPARATION (4%) o 1.35mL of acrylamide stock solution is added to 1mL of stacking gel buffer of pH 6.8 and 7.5mL of distilled water. o Then to this mixture, add 5%APS 50µL 10%SDS 0.1mL TEMED 10µL o Remove the water from the top of resolving gel and pour the stacking gel mix and place the comb in stacking gel and allow to set.  After the formation of the gel, the comb is removed carefully.  Sample mixed with sample buffer was loaded in wells.  The electrode were connected and buffer was poured in tank.  After connection, the power pack was turned on.  After the sample reached the lower edge, power pack was turned off.  Gel was removed from plates and kept overnight in staining solution.  Next day, the gel was removed from staining solution and kept in destaining solution.  The protein bands only retains the colour.RESULT: SDS PAGE was performed. 18
  18. 18. MOLECULAR MODELLING Molecular modeling exercise was carried out using the ball &stick method madeavailable through the modeling kit (1.tarson 2.polylabs molecular modeling kits) supplied tothe students. Each student constructed the model of a compound whose structure is asdepicted in the record work. Constructions were carried out using color coded kit components that specificallyidentified the nature and position of each element within a compound’s chemical structure. C-C, C-N, and C-X (where X is any element) bond lengths and bond angles weremeasured using centimeter scale and the values were noted. The objective of the exercisewas to learn the possibility of constructing chemical structures in relation to their elementalorientations and positioning within a given compound using constructional materials thatcould then be extrapolated to construct and understand similar or more complex chemicalstructures through the use of computer programs /bioinformatics tools.RESULT: The compound constructed is ……………… 19

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