protein estimation teaching kit manual

Document Sample
protein estimation teaching kit manual Powered By Docstoc
					Protein Estimation        GeNeiTM   Protein Estimation                         GeNeiTM




                                    GeNeiTM Protein Estimation
                                          Teaching Kit
                                             Manual




                                                 Cat No.         New Cat No.
                                                   KT42             106193
                                                     Revision No.: 00010905
© Bangalore Genei, 2007             © Bangalore Genei, 2007
Protein Estimation        GeNeiTM   Protein Estimation                         GeNeiTM

                                                              CONTENTS

                                                                         Page No.

                                                 Objective                 3
                                                 Kit Description           3
                                           Bradford’s method
                                                 Principle                 4
                                                 Materials Provided        4
                                                 Procedure                 5
                                                 Result                    6
                                           Biuret’s method
                                                 Principle                 7
                                                 Materials Provided        7
                                                 Procedure                 8
                                                 Result                    9
                                           Lowry’s method
                                                 Principle                10
                                                 Materials Provided       10
                                                 Procedure                11
                                                 Result                   12
                                                 Ordering Information     13

© Bangalore Genei, 2007             © Bangalore Genei, 2007        1
Protein Estimation            GeNeiTM   Protein Estimation                                 GeNeiTM

                                        Objective:
                                             To learn three commonly used Protein estimation
                                        methods:
                                         • Bradford method
                                         • Biuret method
                                         • Lowry’s method

                                        Kit Description:
                                             The kit contains BSA as the protein standard, using which
                                        standard curves will be constructed by three different methods.
                                        These curves will then be used to estimate the concentration
                                        of protein in test samples supplied.

                                            KT42    :   The kit is designed to estimate protein
                                                        concentration by three different methods.
                                                        5 experiments can be carried out with each
                                                        method.

                                        Duration of experiment: All three methods of protein
                                        estimation can be carried out simultaneously or independently.
                                        Approximate time taken for each method is indicated below:

                                            •   Bradford Method: 1 hour
                                            •   Biuret Method : 1 hour
                                            •   Lowry’s Method : 2 hours

                                        Materials Required:
                                        Equipment : Spectrophotometer/colorimeter.
                                        Glassware : Conical flask, Measuring cylinder,
                                                        Test tubes.
                                        Reagent : Distilled water.
                                        Other Requirements : Glass cuvette, Micropipette, Tips.
© Bangalore Genei, 2007   2             © Bangalore Genei, 2007     3
Protein Estimation                                   GeNeiTM       Protein Estimation                                  GeNeiTM

I. Bradford’s method of Protein Estimation:                        Note:
                                                                     •   Read the entire procedure before starting the
Principle:                                                               experiment.
    Bradford’s assay is a rapid and accurate method for              •   Do not reconstitute all test samples/standard vials at a
estimation of protein concentration. When compared to                    time, unless all 5 experiments are carried out within
Lowry’s Method, it is subjected to less interference by                  2 weeks.
common reagents and non-protein components of biological             •   Test samples provided are of different concentrations.
sample.                                                              •   Reconstitute a vial of BSA with 1.5 ml of distilled water
                                                                         to get a stock concentration of 0.5 mg/ml and store at
    The assay relies on the binding of the dye Coomassie blue            4°C. Use this as a standard for two experiments within
G250 to protein. The quantity of protein can be estimated by             2 weeks of reconstitution.
determining the amount of dye in the blue ionic form. This is        •   Reconstitute a vial of test sample with 0.5 ml of distilled
usually achieved by measuring the absorbance of the solution             water and store at 4°C. Use this as test for two
at 595 nm or 625 nm.                                                     experiments within 2 weeks of reconstitution.

Materials Provided:                                                Procedure:
   The list below provides information about the materials           1. Pipette standard BSA (0.5 mg/ml) and test sample as
supplied in the kit. The products should be stored as suggested.        indicated in table 1, in duplicates.
Use the kit within 6 months of arrival.                              2. Adjust the volume to 0.2 ml with distilled water.
                                                                     3. Add 3 ml of Bradford’s reagent and mix it thoroughly.
                                   Quantity                             Incubate at room temperature for 10 minutes.
       Materials                                      Store          4. Read the optical density on spectrophotometer at
                                   (5 expts.)                           595 nm/625 nm or on a colorimeter using a suitable
BSA (Protein standard)             3 x 0.75 mg         4°C              filter and record the readings.
Bradford’s reagent                     250 ml          4°C
Test samples (A, B & C)                0.5 ml each     4°C




© Bangalore Genei, 2007        4                                   © Bangalore Genei, 2007        5
Protein Estimation                                  GeNeiTM      Protein Estimation                                   GeNeiTM

 Sl.      Std.    Amount     Water        OD      Average OD     II. Biuret’s method of Protein Estimation:
 No.    (in µg)    (in µl)   (in µl)    Reading    Reading
 1      Blank        0        200
                                                                 Principle:
                                                                      Biuret method is the most simple method for protein
 2        10         20       180                                estimation. This method is sensitive to the amino acid
 3        20         40       160                                composition of the protein. Its sensitivity is moderately constant
                                                                 from protein to protein and because of its simple procedure
 4        40         80       120                                and quick result, it is used to estimate protein in crude extract
 5        60        120        80                                over a large range of concentration. This method can also be
                                                                 used to monitor the concentration of protein during its
 6        80        160        40                                purification.
 7     Sample        50       150
                                                                     This assay is based on copper ions binding to peptide bonds
                                                                 of protein under alkaline conditions to give a violet or purple
Table 1: OD readings - Bradford Method                           colour. The intensity of the charge - transfer absorption bond
                                                                 resulting from the Cu-protein complex is linearly proportional
  5. Construct a calibration or standard curve by plotting       to the mass of protein present in the solution. The
     average optical density reading on ‘Y’ axis against         chromophore or light absorbing center seems to be a complex
     standard protein concentration (in µg) on ‘X’ axis.         between the peptide backbone and cupric ions.
  6. Record the value ‘x’ from the graph corresponding to
     the optical density reading for the test sample.            Materials Provided:
  7. Calculate the concentration of protein in the test sample      The list below provides information about the materials
     using the following formula:                                supplied in the kit. The products should be stored as
                                                                 suggested. Use the kit within 6 months of arrival.
       Protein concentration in test sample = x/v mg/ml.
       Where      x - Value from graph in µg                                                        Quantity
                  v - Volume of sample in µl.                           Materials                                        Store
                                                                                                    (5 expts.)
Result:                                                          BSA (Protein standard)            3 x 75 mg              4°C
   From the standard curve, determine and report                 5X Biuret reagent                     50 ml              4°C
concentration of protein in the test sample.                     Biuret reagent diluent                200 ml             4°C
                                                                 Test samples (A, B & C)               0.5 ml each        4°C


© Bangalore Genei, 2007             6                            © Bangalore Genei, 2007           7
Protein Estimation                                  GeNeiTM         Protein Estimation                                  GeNeiTM

Note:                                                               Sl.   Std.  Amount          Water       OD        Average OD
  •   Read the entire procedure before starting the                 No. (in mg) (in µl)         (in µl)   Reading      Reading
      experiment.                                                   1       Blank         0      200
  •   Do not reconstitute all test samples/standard vials at a
      time, unless all 5 experiments are carried out within         2         1.0        20      180
      2 weeks.                                                      3         2.0        40      160
  •   Test samples provided are of different concentrations.
  •   Reconstitute a vial of BSA with 1.5 ml of distilled water     4         4.0        80      120
      to get a stock concentration of 50 mg/ml and store at         5         8.0       160       40
      4°C. Use this as a standard for two experiments within
      2 weeks of reconstitution.                                    6        10.0       200         0
  •   Reconstitute a vial of test sample with 0.5 ml of distilled   7      Sample        50      150
      water and store at 4°C. Use this as test for two
      experiments within 2 weeks of reconstitution.
  •   To prepare Biuret reagent: Mix one volume of Biuret           Table 2: OD readings - Biuret Method.
      reagent and 4 volumes of Biuret reagent diluent, before
      use.                                                              5. Construct a calibration curve by plotting average optical
                                                                           density reading on ‘Y’ axis against standard protein
                                                                           concentration (in mg) on ‘X’ axis.
Procedure:
                                                                        6. Record the value ‘x’ from the graph corresponding to the
  1. Pipette standard BSA (50 mg/ml) and test sample as                    optical density reading for the test sample.
     indicated in table 2, in duplicates.                               7. Calculate the sample concentration using the following
  2. Adjust the volume to 0.2 ml with distilled water.                     formula:
  3. Add 3 ml of Biuret reagent, mix and incubate at room
     temperature for 10 minutes.                                           Protein concentration in test sample
  4. Read the optical density using spectrophotometer at                                       = (x/v) x 1000 mg/ml.
     600 nm or colorimeter using a suitable filter and record
     the readings.
                                                                           Where        x - Value from graph in mg
                                                                                        v - Volume of sample in µl.

                                                                    Result:
                                                                       From the standard curve, determine and report
                                                                    concentration of protein in the test sample.

© Bangalore Genei, 2007         8                                   © Bangalore Genei, 2007          9
Protein Estimation                                        GeNeiTM   Protein Estimation                                  GeNeiTM

III. Lowry’s method of Protein Estimation:                          Note:
                                                                      •   Read the entire procedure before starting the
Principle:                                                                experiment.
    Lowry’s method of protein estimation is the most widely           •   Do not reconstitute all test samples/standard vials at a
accepted method for accurate determination of protein                     time, unless all 5 experiments are carried out within
concentration. The method is a combination of Biuret reaction,            2 weeks.
and Folin-Ciocalteau reaction. In the first step of the reaction,     •   Test samples provided are of different concentrations.
protein binds to copper in alkaline medium and produces               •   Reconstitute a vial of BSA with 1.5 ml of distilled water
Cu2+. (cupric ion). In the second step, Cu2+ catalyzes oxidation          to get a stock concentration of 0.5 mg/ml and store at
of     aromatic          amino      acids      by      reducing           4°C. Use this as a standard for two experiments within
phosphomolybdotungstate to heteropolymolybdenum blue.                     2 weeks of reconstitution.
This reaction produces strong blue colour, which                      •   Reconstitute a vial of test sample with 0.5 ml of distilled
predominantly depends upon tyrosine and tryptophan content                water and store at 4°C. Use this as test for two
of protein and to a lesser extent cysteine and other residues             experiments within 2 weeks of reconstitution.
in protein.                                                           •   To prepare complex forming solution: Mix 0.5 ml of
                                                                          solution I and 50 ml of solution II, just before use.
Materials Provided:
   The list below provides information about the materials          Procedure:
supplied in the kit. The products should be stored as suggested.      1. Pipette standard BSA (0.5 mg/ml) and test sample as
Use the kit within 6 months of arrival.                                  given in table 3, in duplicates.
                                                                      2. Adjust the volume to 0.2 ml with distilled water.
                                          Quantity                    3. Add 3 ml of complex forming reagent, mix and keep for
          Materials                                        Store         10 minutes.
                                          (5 expts.)
                                                                      4. Add 0.3 ml of Solution III, mix thoroughly and incubate
 BSA (Protein standard)                 3 x 0.75 mg         4°C          for 20 - 30 minutes at room temperature.
 Solution I: Copper solution                  3 ml          4°C
 Solution II: Alkaline tartarate           250 ml           4°C
 Solution III: Folin’s reagent               30 ml          4°C
 Test samples (A, B & C)                    0.5 ml each     4°C




© Bangalore Genei, 2007            10                               © Bangalore Genei, 2007        11
Protein Estimation                                  GeNeiTM         Protein Estimation                         GeNeiTM

    5. Read the optical density on spectrophotometer at 660nm       Ordering Information
       or on a colorimeter using suitable filter and record the
       readings.
                                                                    Product                          Size      Cat #
Sl.       Std.    Amount     Water       OD      Average OD
No.     (in µg)    (in µl)   (in µl)   Reading    Reading           GeNeiTM Protein Estimation        1 Pack   KT42
                                                                    Teaching Kit
1       Blank        0        200                                   (Consumables for 3 x 5 experiments)
2         10        20        180
3         20        40        160
4         40        80        120
                                                                    Email:
5         60        120        80                                   Sales:
                                                                    geneisales@sanmargroup.com
6         80        160        40
7      Sample       50        150                                   Customer Support:
                                                                    geneitechsupport@sanmargroup.com
Table 3: OD readings - Lowry’s Method
    6. Plot a calibration curve by taking optical density reading
       on ‘Y’ axis against standard protein concentration
       (in µg) on ‘X’ axis.
    7. Record the value ‘x’ from the graph corresponding to
       the optical density reading for the test sample.
    8. Calculate the protein concentration using the following
       formula:

      Protein concentration in test sample = x/v mg/ml.
      Where: x - Value from graph in µg
               v - Volume of sample in µl.

Result:
       From the standard curve, determine and report
concentration of protein in the test sample.
© Bangalore Genei, 2007       12                                    © Bangalore Genei, 2007   13
Protein Estimation             GeNeiTM   Protein Estimation        GeNeiTM

Notes:




© Bangalore Genei, 2007   14             © Bangalore Genei, 2007

				
DOCUMENT INFO
Shared By:
Stats:
views:39
posted:1/7/2013
language:Unknown
pages:9