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Affinity Chromatography and SDS PAGE gel

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					                     Affinity Chromatography and SDS-PAGE

Introduction
       Often one of the goals in biochemistry and molecular biology is the isolation of a
single pure protein from a complex mixture of proteins.       Most fractionation procedures
involve many separations. To monitor such protein isolation, electrophoresis is the method
of choice. Even though electrophoresis can be considered a type of fractionation in its own
right, another popular technique is chromatography.      Here, proteins are separated from
each other as they are passed through and interact with a matrix. No electric field is used
here, but the fractionation procedure relies on the flow of the solvent to aid in separation.
       There are many types of chromatographic matrices available commercially; each one
is designed to exploit the physical and chemical differences that exist among proteins. In
gel filtration chromatography, matrix beads of different sizes are used to separate
proteins based upon molecular size.      With this technique, larger proteins pass readily
between beads whereas smaller proteins are retarded in their passage through the matrix
as they pass through holes in the beads. Gel filtration chromatography is most useful when
attempting to separate proteins that differ           greatly in size.      In ion-exchange
chromatography, differences in protein native charge are used as the basis of separation.
Proteins that are positively charged at a particular pH will be attracted to negatively
charged matrix beads, whereas the negatively charged beads will repel negatively charged
proteins. The reverse situation is also true using positively charged beads. However, one of
the main disadvantages of both of these chromatographic procedures is that neither
technique will isolate a single protein. For example, gel filtration separates proteins that
are similar in size from those that are dissimilar.    With this technique, it is not really
possible to separate those proteins that are similar in size. Ion exchange chromatography
has the same limitations since proteins that have similar charges cannot be separated from
each other.
       Another chromatographic technique called affinity chromatography is capable of
separating a single protein from a complex mixture of proteins. It does so because it takes
advantage of the interaction of one molecule with a second molecule (ligand). Many types of
molecules can be separated using affinity chromatography. The following table lists just a
few of these molecules.
                 Substance isolated by
                 Affinity Chromatography            Ligand

                 Enzyme                         Substrate or Cofactor
                 Antibody                       Antigen, Virus, Cell
                 Polysaccharide, glycoprotein   Lectin
                 Nucleic acid binding protein   Nucleic acid
                 Hormone receptor               Hormone


          The basic procedure for affinity chromatography is outlined in Figure 1. The first

step involves the covalent binding of the complementary binding molecule or ligand to the

matrix, usually small agarose beads. The matrix is then poured into a column and allowed to

settle. The column is then washed several times to remove any impurities and to equilibrate

the column with the appropriate buffer. The mixture of proteins is then applied to the

column and allowed to interact with the matrix beads as the solution moves through the

column by gravity. During this time, the protein to be isolated will bind specifically to the

ligand on the beads while the remaining proteins will pass through the column. The column is

then washed with buffer to remove any proteins that are not specifically bound to the

column.    Finally, the protein bound to the column is then eluted from the column under

specific conditions that will disrupt the interaction of the protein with the ligand. The

solution containing the protein is collected in a tube as it drips from the column. The final

step involves some type of assessment of the chromatography to make sure that the protein

is pure. As mentioned previously, electrophoresis is often used.

          In this experiment, you will be isolating horse serum albumin from diluted horse
serum by affinity chromatography. The matrix in the column will be Affi-Gel blue, which
contains a reactive blue dye molecule (Cibacron Blue F3GA) covalently linked to agarose
beads. To elute the albumin from the column, a 1% solution of SDS will be used. You will
then assess the chromatographic run by subjecting your sample of albumin, the column flow-
through material (all other serum proteins), and the diluted horse serum to SDS-PAGE
following by staining with Coomassie blue.          The following standard proteins will be
electrophoresed with your samples:




                                                2
                                  Molecular Weight
Standard Protein                    (in Daltons)


Bovine serum albumin, dimer          132,000
Bovine serum albumin, monomer         66,000
Ovalbumin                             43,000
Myoglobin                             17,000




                              3
Procedure:
Preparation of the Affi-Gel Affinity Column


1. Secure your column in a vertical position on a ring stand such that the narrow, plugged
     end of the column is facing downward. The porous disc in the bottom of the column
     ensures that the matrix beads stay in the column, but allows solutions and proteins to
     flow freely through the column.
2. Place a small beaker under the column to catch the buffer and any flow-through. Since
     the Affi-Gel blue is supplied as a slurry of agarose beads in a buffer, mix the slurry
     thoroughly to make sure that the solution is suspended uniformly. Quickly transfer 2 mL
     of the slurry to your column. As the Affi-Gel blue is settling, you will notice two layers
     forming: a bottom layer of Affi-Gel blue and a top layer of buffer. Allow this to settle
     for several minutes.
3. Carefully remove the blue plug at the bottom of the column. Buffer should begin to flow
     dropwise into the beaker below. When the level of the buffer approaches the top of the
     Affi-Gel blue layer, fill the column with column buffer (0.15 M NaCl, 10 mM Tris, pH 8.0)
     and allow the solution to flow through the column. Repeat this step. NEVER ALLOW
     THE AFFI-GEL TO BECOME DRY. If necessary, add more column buffer to prevent it
     from becoming dry.


Applying the Horse Serum


1. Obtain five small microcentrifuge tubes and label tubes #1, #2, #3, flow through, and
     bound. Pipet 10 L of the diluted horse serum in tube #1.
2. After the column has been washed twice, allow the buffer to drain to within 1 or 2 mm of
     the top of the Affi-Gel blue. Carefully pipet 500 L of the horse serum into the column
     and immediately place the tube marked “flow through” under the column and collect the
     solution that contains the horse serum minus those proteins bound to the column.
3.    When the sample has been collected, remove the tube “flow through” and wash the
     column twice with column buffer.
4. Allow the final wash to drain to within several mm of the top of the matrix. Pipet 1 mL
     of elution buffer (1% SDS) into the column and immediately place the tube marked
     “bound” under the column and collect the solution that contains the protein bound to the
     column.




                                               4
Sample Preparation for SDS-PAGE


1. Add 10 L of distilled water to the 10 L serum sample in tube #1.
2. Pipet 20 L of the contents of the tube labeled “flow through” to tube #2.
3. Pipet 20 L of the contents of the tube labeled “bound” into tube #3.
4. Add 20 L of SDS-PAGE sample buffer to each of the tubes labeled #1, #2, and #3.
     Mix thoroughly. Boil tubes for 5 minutes in a boiling water bath.
5. Load 10 L of each of the samples (#1-#3) into separate lanes of an SDS-PAGE gel. Also
     load 10 L of the standard proteins in an adjacent well.
6. Electrophorese the samples and stain the gels overnight in Coomassie blue as detailed in
     the previous laboratory exercise.
7. Obtain a digital picture of your stained gel for your lab report. Label each lane plus all
     four standards with molecular weights, and title your drawing “Figure 1. SDS-PAGE of
     affinity chromatography procedure used to isolate albumin from horse serum.”


Questions to Answer:


1.   Describe the results of your albumin purification including a description of the relative
     mobility of the purified protein. Did your affinity chromatography column remove all of
     the albumin from the serum? How do you know? How pure was the affinity-isolated
     protein?


2. An antiserum contains hundreds of proteins in addition to antibodies and is therefore a
     complex, impure mixture of proteins. You have been asked by the Centers for Disease
     Control and Prevention to test people to determine if they have been exposed to the
     bovine spongiform encephalopathy virus that causes mad cow disease. Design an affinity
     chromatography experiment to purify antibodies that react with thevirus from serum
     you have collected from these people. Be specific in your methodology. Would either
     influenza virus or respiratory syncycial virus (RSV) serve as a good ligand in this
     experiment? Why?


3. In the chromatography experiment you performed in lab, the serum albumin was eluted
     from the column using 1% SDS.         What, specifically, did this detergent do to the
     albumin-ligand interaction?    If you were interested in isolating albumin in its native
     conformation, would you elute the albumin from the column with SDS? If not, what



                                                5
   other method could you use to elute the albumin in this experiment and still preserve its
   structure?


What to turn in:
Figure 1, questions and answers to questions




                                               6
1. Couple ligand to matrix covalently.




         Matrix                ligand
         beads



2. Apply sample containing protein plus impurities and wash away impurities.




                        Protein
                        (antibody)      impurities



3. Elute pure protein



                          +    elution buffer

                                                                               Purified
                                                                               protein




            Figure 1. General principles of affinity chromatography.




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Description: Affinity Chromatography and SDS PAGE gel