Protein purification tutorial by techmaster

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									Protein Purification Tutorial


              Protein purification tutorial
                                TITLE PAGE
                                   graphic




       "Proteins" a word derived from the Greek word, Proteios,
       which means "of the first rank" was coined by Jons J.
       Berzelius in 1938 to stress the importance of this group of
       organic compounds. Proteins have been described as the
       servants of life. Most proteins are enzymes but in addition
       there are also regulatory and structural proteins.
  information in these genomes has dramatically                        can identify the gene within
Protein Purification Tutorial the changed research. If youthe encoded–Why purifythe the
  genomic DNA sequence, you already know        amino acid sequence of
                                                                            Intro
                                                                                    protein and
                                                                                                 proteins?
    regulatory elements that control the expression of this gene. You don’t, however, know the structural
    organization of the protein or how it is regulated. If you do not have a genomic database or cannot identify
    the protein coding region within the genome, you may want to purify the protein in order to help you to isolate
    the gene.
      – 1) If you have not yet isolated the gene that encodes the protein, you may want to purify the protein for
         any of the following reasons:
      – The purified protein can be used to determine the amino acid sequence. The sequence can then be
         used as a probe to help in the isolation of the gene.
      – The purified protein can be used to produce antibodies that can be used as a probe to help in the
         isolation of the gene.
      – The purified protein can be analyzed by mass spectroscopy. The molecular weight can then be used
         as a screen of the genomic sequence for the gene.
      – 2) If you have already isolated the gene that encodes the protein, you may want to purify the protein for
         any of the following reasons:
      – Pure proteins are required for structural analysis.(x-ray crystallography and NMR spectroscopy)(Figure
         1).
      – Pure proteins are required to study enzyme function.
      – Pure proteins are needed in order to learn about what other proteins or nucleic acids they interacts with.
         (Figure 2)
      – Pure proteins are needed for studies of enzyme regulation (are their regulatory subunits, is it regulated
         by phosphorylation, is the protein regulated by interaction with other proteins.)
Protein Purification Tutorial     Objectives




   • In this tutorial you will:
       –   1
       –   2
       –   3
       –   4
       –   5
Protein Purification Tutorial                                           Intro – table of common techniques



       Table of common methods of protein purification
                                                                    •     Purification procedures attempt
           Property                    Methods                            maintain the protein in native fo
                                                                          Although some proteins can be
             solubility                Precipitation with
                                       ammonium sulfate
                                                                          natured, most cannot!
                                       (salting out)*               •     To purify a protein from a mixtu
           Size / shape                Size-exclusion                     biochemists exploit the ways th
                                       chromotography                     individual proteins differ from o
                                                                          another. They differ in:
           Isoelectricpoint            Ion exhange
           (charge)                    chromatography                      – Thermal stability*

           binding to small            Affinity
           molecules                   chromatography




        *Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes.
        It is commonly one of the first steps in a purification scheme.
                                                                  *For most protein purifications, all steps
                                                                  are carried out at ~5°C to slow down
                                                                  degradative processes.
Protein Purification Tutorial                                                                             Intro side bar




   Picture of protein gel with lanes
      showing sequential purification
      steps


                        Purification Table

                       Procedure          Fraction vol   Total Prot    Activity       Specific activity   Purificati Yield
                                          (ml)           (mg)          (units)        Units/mg            on factor
                       Crude cellular     1400           10000         100000         10                  1        100%
                       extract
                       Size-exclusion     90             400           80000          200                 20     80%

                       Ion exchange       80             100           60,000         600                 3         75%




                                        Add row with ammonium sulfate data. Include two
                                        colums at end called Purification factor and Yield.
                            Note: The type and order of steps are customized for each
                            protein to be purified. An effective purification step results in
                            a high yield (minimal loss of enzyme activity) and large
                            purification factor (large increase in specific activity).
                                                                     Intro – flow chart. Purification is a multi step
Protein Purification Tutorial                                                                             procedure

   Purification is a multi-step procedure.

    Sample
                                    Separation
                                    technique


                                                                   Repeat with another
                Fractionation
                                                                            separation
                                                                   technique until pure


                                                                                                              No
                                Assay total protein
                                Assay enzyme activity




                    No                                  yes   Combine                                  Pure?
    Set aside                    Is there activity?                              Monitor purity
                                                              Fractions


                                                                                                            yes



                                                                                           Prepare for analytical technique
Protein Purification Tutorial                 Intro – first steps.



 •   First steps
      – 1. Source. A good source is
        cheap and readily available.
        Many proteins are enriched in
        specific tissues, for example
        hemoglobin in blood. For this
        reason, these tissues may be
        excellent sources for your protein.
      – 2. Assay: Most assays are
        chemical reactions catalyzed by
        specific enzymatic activities.
        Proteins that have no activity are
        usually assayed using SDS
        polyacrylamide gels.
Protein Purification Tutorial                Assay – develop an assay




   •   First steps – Develop an Assay

   1. An assay for an enzyme is a
      method for quantifying its activity.

   Since the assay is repeated many
      times, it is important that it be a
      simple procedure. Usually
      enzyme activity is monitored as a
      change in absorbance which can
      be measured using a
      spectrophotometer. For example
      an assay for ribonuclease
      measures the change in
      absorbance that accompanies the
      breakdown of RNA to
      ribonucleotides.
Protein Purification Tutorial           Intro – Preparing the sample (Crude Extract)


  First steps: Preparing the sample – Crude extract.
   Protein from cells or tissue



                                                                     Supernatant with
                                                                     Soluble protein
                     Break cells,
                     tissue, or organ

                     Blender,
                     homogenizer,
                     sonication,
                     pressure,
   Microbial cells   psmotic                             Pellet with intact cells, organelles, mem
   or tissue
Protein Purification Tutorial                     Segway to Chromotography




   •   Some kind of graphic.    •   Note: In order to isolate sufficient
                                    quantities of protein, you may
                                    need to start with kilogram
                                    quantities of source tissues.
                                    These amounts can best be
                                    handled using precipitation
                                    methods (e.g. ammonium sulfate
                                    precipitation). Later in the
                                    purification, large columns can be
                                    used to handle gram to milligram
                                    quantities. Amounts handled on
                                    gels are typically in microgram
                                    quantities.
                                                                     Intro – flow chart. Purification is a multi step
Protein Purification Tutorial                                                                             procedure

   Purification is a multi-step procedure.

    Sample
                                    Separation
                                    technique


                                                                   Repeat with another
                Fractionation
                                                                            separation
                                                                   technique until pure


                                                                                                              No
                                Assay total protein
                                Assay enzyme activity




                    No                                  yes   Combine                                  Pure?
    Set aside                    Is there activity?                              Monitor purity
                                                              Fractions


                                                                                                            yes



                                                                                           Prepare for analytical technique
Protein Purification Tutorial                            Over view of the apparatus

Column Chromotography –general case
                                      –   Glass column
                                      –   Reservoir
                                      –   Solid matrix – beads
                                      –   Solution of protein.
                                      –   Effluent.
                                      –   Other terms?


                                We think it would be better to have a resevoir
                                attached to a cap on the top of the column via a
                                plastic tube, show minimal liquid on top of the
                                column bed, show a tube coming out of the
                                bottom of the column that has a clamp of
                                stopcock. Perhaps a schematic on the left and a
                                photo of an actual column running in a cold box
                                over a fraction collector on the right.
                                Should emphasize that the basic set=up is the
                                same for all column types, but the
                                characteristics of the beads vary.
Protein Purification Tutorial                                Load sample (4 protein mix)




   •   Image of apparatus with protein     •   TEXT. The crude extract is
       mixture layered on top                  placed on top of the solid matrix.
                                               (In this case we are using a
                                               mixture of 4 proteins, indicated by
                                               different colors.)

                                           • (As the animation proceeds)
                                           The proteins move at different rates
                                              through the matrix based on the
                                              properties of the proteins and the
                                              type of column beads.

        Note - this should be shown as
        a single band (possibly brown or
        striped with four colors)
Protein Purification Tutorial                                       Collect fractions.




   •   Fractionation            • Text:
                                As the column separates the proteins
                                   in the mixture, the ―effluent‖ drips
                                   into a series of fraction tubes that
                                   are moving at a specific rate of
                                   speed. These tubes are called
                                   fractions.

                                Here we are showing 20 tubes.
                                   Fraction collectors in most labs
                                   have about 75-200 tubes.


                                  Be sure to remove color from
                                  column as it drips into the tubes
                                  below! If the sample is spread over
                                  three tubes, the center tube will be
                                  darker in color.
Protein Purification Tutorial                                          --3 questions—




   •   In reality, proteins aren’t color-   1. How do we know which fractions
       coded so we must ask ourselves          contain protein?
       these 3 questions:
                                            2. Which of those fractions contain
                                               the desired protein?

                                            3. How do we assess the purity?




       ???
Protein Purification Tutorial                                             Question 1 – take A280 Screen 1.

      Question 1. How do we know which fractions contain protein?
                                                              •   Total protein a can be estimated
                                                                  by taking the absorbance at 280
                                                                  nm in a spectrophotometer.
                                                                  Aromatic amino acids absorb light
                                                                  at this wavelength causing all
                                                                           to have
                                                                  proteins Take A280 absorbance at
Fraction
#
         1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20       280nm. Many fraction collectors
                                                                  measure the A280 as the column
                                                                  is running.
Protein Purification Tutorial                                            Question 1 – take A280 Screen 2 .

       Question 1. How do we know which fractions contain protein?
                                                              •   Total protein a can be estimated
                                                                  by taking the absorbance at 280
                                                                  nm in a spectrophotometer.
                                                                  Aromatic amino acids absorb light
                                                                  at this wavelength causing all
Fraction
                                                                  proteins to have absorbance at
         1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#                                                                 280nm. Many fraction collectors
       0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0                    measure the A280 as the column
A280                                                              is running.
                                                              •   The A280values can be plotted
                                                                          Plot values
                                                                  against the fraction number in is
                                                                  what is called an elution profile.
Protein Purification Tutorial                                            Question 1 – take A280 Screen 3 .

       Question 1. How do we know which fractions contain protein?
                                                              •   Total protein a can be estimated
                                                                  by taking the absorbance at 280
                                                                  nm in a spectrophotometer.
                                                              •   The values can be plotted against
                                                                  the fraction number in is what is
Fraction                                                          called an elution profile.
#
         1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
       0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280                                                          •   Notice the peaks on the graph.
                                                                  These indicate where the fractions
                         Peaks                                    are that contain protein.



A280




                  Fraction #
Protein Purification Tutorial                                         Question 1 – take A280 Screen 4 .

       Question 1. How do we know which fractions contain protein?
                                                          •   Total protein a can be estimated
                                                              by taking the absorbance at 280
                                                              nm in a spectrophotometer.
                                                          •   The values can be plotted against
                                                              the fraction number in is what is
Fraction                                                      called an elution profile.
#
         1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
       0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280                                                      •   Notice the peaks on the graph.
                                                              These indicate where the fractions
                         Peaks                                are that contain protein.



A280




                  Fraction #
Protein Purification Tutorial                                        Question 2 – take A280 Screen 1 .

       Question 2. Which fractions contained the desired protein?
                                                          •   Enzyme activity can be
                                                              determined by performing an
                                                              enzyme assay on each fraction
                                                              that contains protein.

Fraction
         1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
# Fraction
  #
         0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280
                                                                    Enz. Assay.



A280




                  Fraction #
 Protein Purification Tutorial                                             Question 2 – take A280 Screen 2.

        Question 2. Which fractions contained the desired enzyme?
                                                               •   Enzyme activity can be
                                                                   determined by performing an
                                                                   enzyme assay on each fraction
                                                                   that contains protein.

 Fraction
          1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20   •   Notice the results of the enzyme
 #
                                                                   assay. The highest activity
           0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280                                                               corresponds to one of the peaks.
EnzAssay
Results
            Need to substitute values for the colored
            spots since we are switching to an absorbance               Now we can have them discard
            based assay.                                                tubes that don’t have enzyme
                                                                        activity.

 A280




                        Fraction #
 Protein Purification Tutorial                                             Question 2 – take A280 Screen 3.

        Question 2. Which fractions contained the desired protein?
                                                               •   Enzyme activity can be
                                                                   determined by performing an
                                                                   enzyme assay on each fraction
                                                                   that contains protein.

 Fraction
          1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20   •   Notice the results of the enzyme
 #
                                                                   assay. Notice that the highest
           0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280                                                               activity corresponds to one of the
                                                                   peaks.
EnzAssay
Results




 A280                                                          •   Discard the fractions that don’t
                                                                   contain protein by clicking on the
                                                                   tubes that don’t contain protein.



                       Fraction #
Protein Purification Tutorial                                                    POOL fractions – screen 1

      Combine (pool) the fractions with activity.

                                                                  1. NEXT We want to pool the
                                                                     fractions that have enzyme
                                                                     activity.


Fraction
#
         1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20


       It may be useful to consider more than just the activity of a
       fraction. Specific activity is a measure of the amount of
       enzyme activity per amount of protein (units/mg). The
       higher the specific activity, the higher the purity. When
       pooling fractions, judgement is needed as to whether to
       optimize yield or specific activity.                                    Pool
                                                                             fractions




                       Fraction #
Protein Purification Tutorial                          POOL fractions – screen 2

    Combine (pool) the fractions with protein and activity.

                                       1. The fractions are pooled together.

                                       How do we monitor the progress of
                                         the purification?




              Fraction #
Protein Purification Tutorial                                                                         Assess purity – screen 1

                                                           1. Look at it on a gel. Even when
         Question 3: Is this pooled sample pure? How do you monitor the progress? one band is
                                                              overloaded, if only
                                                                                   visible, it is likely to be pure and a
       Standards | Crude Ext. | Pooled fractions
                                                                                   monomer, a homo-dimer, or a
                                                                                   homo-multimer. If not a single
                                                                                   band, additional bands that purify
                                                                                   in parallel, will remain proportional
                                                                                   throughout the purification. If a
Purification Table                                                                 band co-purifies, it is likely to be a
                                           Purification Table
                                                                                   subunit for the enzyme.
                                           Procedure            Fraction vol   2. Calculate the specific activity by
                                                                               Total Prot       Activity       Specific activity
                                                                (ml)           (mg)doing a careful quantitative assay
                                                                                                (units)        Units/mg

                                           Crude cellular       1400               for enzyme activity/total protein.
                                                                               10000            100000         10
                                           extract
                                           Separation           90              400                80000             200
                                           method 1



                                            Results:
                                            1.     Gel shows more than one band.




                                        Since the sample is not pure, you must pass pooled sample through another separation technique

                                                                                           Another separation
Protein Purification Tutorial                                                                     Assess purity – screen 2

         Question 3: Is this pooled sample pure? How do you monitor the progress?

       Standards | Crude Ext. | Pooled fractions                               1. Look at it on a gel. A monomer
                                                                                  should have one band.
                                                                               2. Calculate the specific activity by
                                                                                  doing a careful quantitative assay
                                                                                  for enzyme activity/total protein.
Purification Table                         Purification Table
                                            Procedure           Fraction vol     Total Prot   Activity      Specific activity
                                                                (ml)             (mg)         (units)       Units/mg

                                            Crude cellular      1400             10000        100000        10
                                            extract
                                            Separation          90               400          80000         200
                                            method 1
                                            Separation          8                4            60000         15000
                                            method 2


                                            Results:
                                            1.     Gel shows one band
                                            2.     Specific activity is 15000. Looks good



                                        Your protein seems pure. YOU’RE DONE!!!.
Protein Purification Tutorial                                        Intro – table of common techniques



       Table of common methods of protein purification

           Property                   Methods

             solubility               Precipitation with
                                      ammonium sulfate
                                      (salting out)*
                                              The 3 separation techniques of chromotography
           size                       Size-exclusion
                                      chromotography


           Isoelectricpoint           Ion exhange
           (charge)                   chromatography


           binding to small           Affinity
           molecules                  chromatography




        Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes.
        It is commonly one of the first steps in a purification scheme.
Protein Purification Tutorial                                                         Gel filtration 1 -basis


        Here’s our sample mix of proteins.
        Our goal is to purify protein #….
                                                             •   Gel filtration column
                                                                 chromotography separates
                                                                 proteins on the basis of size.

                                                             •   We will start with 4 proteins.
                                                             •   You will want to purify the ―yellow
    60 Kd          20 Kd         20 Kd           5 Kd
                                                                 one‖
    Low pI (6)     Low pI (7)    Medium pI (7)   Hi pI (8)
Protein Purification Tutorial
Protein Purification Tutorial               Gel filtration 2 - close up of beads




                                •   The matrix of a size-exclusion
                                    chromatography column is porous
                                    beads.




                                          Run column




                                        Need two pore sizes (other size
                                        bigger than black proteins, smaller
                                        than existing pores.)
Protein Purification Tutorial          Gel filtration 3 - run close up of column




                                •   The matrix of a gel filtration
                                    column are beads with pores.

                                •   The large green proteins can’t fit
                                    in pores so flows faster.
                                •   The red/yellow medium sized
                                    proteins get trapped in the pores.
                                •   The black small proteins stay
                                    trapped in pores longer.



                                     We’re wondering how this will work in
                                     animation. The black can permeate all
                                     pores and the space between beads, the
                                     yellow and red can permeate the space
                                     between beads and larger pores, the
                                     green will be restricted to the space
                                     between beads.
Protein Purification Tutorial                                 Gel filtration 4 - zoom out




                                   Click on the peak that represents the
                                       protein of the largest molecular
                                       weight?


                                       Be sure to keep in mind that the colors will either
                                       be in the column or in the tubes, not both!




     Tubes march in from left   A280




                                                 Fraction #
Protein Purification Tutorial                                                                    Gel Filtration 5.




   •                                                  •   Many columns are commercially
                                                          made. Here are some examples.
       This could be moved to the earlier view of
       the porous beads.




                                                                 Fig 1.1. Scanning electron micrograph
                                                                 of an agarose gel. Magnification x 50,000.
                                                                 Ref. Anders S. Medin,PhD Thesis,
                                                                 Uppsala University 1995.



                                                    Sephadex./
Protein Purification Tutorial                                                           ION –EXCHANGE 1




                                                          •   Ion-exchange column
                                                              chromotography separates
                                                              proteins on the basis of charge.

                                                          •   We will start with 4 proteins.
                                                          •   pH 7.2
    60 Kd        20 Kd        20 Kd           5 Kd        •   pos charged column
    Low pI (6)   Low pI (7)   Medium pI (7)   Hi pI (8)




                 Need to include a slide on how to determine the charge on a protein,
                 given its pI and the pH.
Protein Purification Tutorial                     Ion Exchange 2 – loaded proteins




                                      •   The matrix of an ion exchange is
                                          positively charged.
                          pos
                                      •   What do you think will happen?

              pos



                                                Run column
                         pos



               pos




                                pos

               pos
Protein Purification Tutorial                             Ion Exchange 3 –column run




                                      •   The matrix of an ion exchange is
                                          positively charged.
                                pos
                                      •   Only the pos charged proteins run
                                          through the pos charged column.
              pos                         The others ―stick‖ to the column.



                         pos
                                           These beads are porous, too, so you can show the
                                           proteins moving right through the beads in the
                                           animation, ifyou like.
               pos




                          pos

               pos
Protein Purification Tutorial                                    Ion Exchange 4- zoom out




                                            Only the POS charged proteins run
                                               through the column.

                                            How can we elute the other proteins?




 20                              1

      Tubes march in from left

                                     A280




                                                    Fraction #
Protein Purification Tutorial                               Ion Exchange 5 - zoom out




                                       Increase the salt.       Add salt




     Tubes march in from left

                                A280




                                              Fraction #
Protein Purification Tutorial                                 Ion Exchange 6 – after salt




                                       Increase the salt.    Add salt
                                       What protein will come of the column
                                          next?
                                                               -         -        ---

                                         Don’t show the charges on the color spots -
                                         students should figure this out on their own!




     Tubes march in from left

                                A280




                                               Fraction #
Protein Purification Tutorial                              Ion Exchange 7 – after salt




                                       Increase the salt.
                                       What protein(s) will come of the
                                          column next? +       ---     ---
                                       Feedback statement.


                                                  Run column




     Tubes march in from left

                                A280




                                              Fraction #
Protein Purification Tutorial                             Ion Exchange 8 – after salt




                                       Red and yellow will have the same
                                         neg charge and will co-elute.




                                                                         1.5



     Tubes march in from left

                                A280                                      Salt
                                                                          concentration




                                                                          0.0


                                             Fraction #
Protein Purification Tutorial            Ion Exchange 9 – Increase salt conc. Again.


                                                              Add salt
                                       Increase the salt concentration




                                                                         1.5



     Tubes march in from left

                                A280                                     Salt
                                                                         concentration




                                                                         0.0


                                              Fraction #
Protein Purification Tutorial               Ion Exchange 10 – rum column prompt.




                                       Run the column.




                                                 Run column



                                                                     1.5



     Tubes march in from left

                                A280                                  Salt
                                                                      concentration




                                                                      0.0


                                             Fraction #
Protein Purification Tutorial                             Ion Exchange 11 – results.




                                       Run the column.




                                                 Run column



                                                                         1.5



     Tubes march in from left

                                A280                                     Salt
                                                                         concentration




                                                                         0.0


                                             Fraction #
Protein Purification Tutorial                              Ion Exchange 12 – results.




                                       Notice that 2 of the proteins eluted at
                                           the same time. Why?
                                       Is our protein pure? We were
                                           supposed to purify the red one

                                       .




                                                                          1.5



     Tubes march in from left

                                A280                                      Salt
                                                                          concentration




                                                                          0.0


                                              Fraction #
Protein Purification Tutorial                      Affinity Chromotography.




   •   Affinity Chromotography   •   See notes below
Protein Purification Tutorial                                                                Monitoring progress.




   •   Monitoring progress.                                            • We need some info on the SDS
                                                                         page and the specific activity.
                                                                       • See Jim’s hand written notes as a
                                                                         guide.




            Place final table here - we can include the thing plot with protein conc going
            down, enzyme amount remaining constant, and specific activity on the rise.

								
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