Chapter 5 Introduction to Studying Proteins by xiaohuicaicai


									   Chapter 5:
 Introduction to
Studying Proteins
• Protein structure and function
  – Molecular structure, levels of structure
• Protein synthesis
  – Post-transcriptional RNA processing (eukaryotic
  – Translation
• Types of proteins
  – Structural proteins
  – Antibodies
  – Enzymes
• Studying proteins
            Proteins in BT/BM
Virtually all BT/BM products involve proteins
in some way . . .
• Whole protein molecules
  – EX: insulin
• Amino acids, short peptides
  – EX: aspartame (two linked amino acids)
• GMOs engineered to produce a protein
  – EX: ―Roundup Ready‖ soybeans that contain an
    added protein for herbicide resistance
• Instruments & equipment used to study or
  synthesize proteins
  – Synthesizers, sequencers, gel apparatus equipment
      In order to work with a protein,
      resesarchers need to know its:
• Amino acid sequence              mkfflllfti gfcwaqyspn tqqgrtsivh lfewrwvdia
                                   lecerylapk gfggvqvspp nenvaihnpf rpwweryqpv
                                   syklctrsgn edefrnmvtr cnnvgvriyv davinhmsgn
  (primary structure)              avsagtsstc gsyfnpgsrd fpavpysgwd fndgkcktgs
                                   gdienyndat qvrdcrlvgl 181 ldlalekdyv rskiaeymnh
                                   lidigvagfr ldaskhmwpg dikaildklh nlnsnwfpag
                                   skpfiyqevi dlggepikss dyfgngrvte fkygaklgtv
• Molecular mass                   irkwngekms ylknwgegwg fmpsdralvf vdnhdnqrgh
                                   gaggasiltf wdarlykmav gfmlahpygf trvmssyrwp
 (reflects protein size, usually   rqfqngndvn dwvgppnnng vikevtinpd ttcgndwvce
 expressed as kilodaltons          hrwrqirnmv nfrnvvdgqp ftnwydngsn qvafgrgnrg
                                   fivfnnddwt fsltlqtglp agtycdvisg dkingnctgi
 (kD))                             kiyvsddgka hfsisnsaed pfiaihaesk l

                                                                55.9 kD
• Three-dimensional

• Also: chemical                                                    (info shown
 behavior (activity,                                                here is for a-
     Protein structure
review … we covered this in Chp. 2

     your book includes it in
       Chp. 5, section 5.1

 we’ll review the material here…
    and add a little as we go
                 Amino acids
• Proteins are very large polymers made from 20
  amino acid monomers.
• There are 20 different R groups—this is the
  variable part of the amino acid and determines its
  specific properties.
• Some R groups are
  hydrophobic (only C & H)
• Some R groups are
• Some R groups are acidic
  and some are basic.
• R groups dictate the
  properties of the
  amino acid
       Some examples of amino

     (also see Table 5.1, p. 133)

 Leucine      Aspartic acid    Glycine      Serine        Lysine
  (Leu)          (Asp)          (Gly)        (Ser)         (Lys)

                 acidic,      simplest;   hydrophilic     basic,
hydrophobic    charged (-)      only H      (polar)     charged (+)
Amino acids are linked together
      by peptide bonds

         +          +
        Protein structure (cont).
• Amino acid monomers are joined together by
  peptide bonds to form a polypeptide (the
  – The amino acid sequence is the primary (1°) structure
    of a protein
• The polypeptide then
  folds up into the
  secondary (2°) and
  tertiary (3°)
  structures of
  the protein
              Secondary structure
• Local structural patterns created by hydrogen
  bonding between amino acids
1) alpha-helix (a-helix)        2) beta-sheet
             Tertiary structure
• Overall 3-D structure of the protein
• Results from:
   Electrostatic interactions:
    (+/- charges interacting) between
    amino acid R groups
   Hydrophobic interactions:
    hydrophobic (nonpolar) amino
    acids moving away from
    hydrophilic (polar) amino acids & water
   Disulfide bonds: strong bonds between
    cysteine amino acids (1 of the 20 types of amino
          Quaternary structure
• Structure of the protein resulting from the
  association of two or more polypeptide
  – Many proteins have >1 subunit and are not
    functional until the quaternary structure has
    been assmbled

             2 subunits              4 subunits
Summary: 4 levels of protein structure

 • Primary structure (1º): the amino
   acid sequence (the ―polypeptide‖).
 • Secondary structure (2º): local
   patterns of folding in the polypeptide.
 • Tertiary structure (3º): the overall,
   3-D shape of the protein.
 • Quaternary structure (4º):
   association of >1 protein subunit.
     4 levels of protein structure

    (1º)                (2º)             (3º)          (4º)
   Primary           Secondary          Tertiary    Quaternary
  Sequence of     Local patterns of      Overall   >1 polypeptide
 amino acids in   folding within the   3-D shape
the polypeptide      polypeptide
• The process in which proteins lose their 3-D
  – Essentially, the loss of tertiary (and usually
    quaternary structure)
  – Primary structure (and possibly some secondary
    structure) is maintained
• Results in loss of function of the protein
• Caused by subjecting proteins to external
  stress, such as heat, strong acids or bases, or
  high concentrations of some chemicals
So how are proteins made?
     “Central dogma” of biology
• Flow of information is from DNA to RNA to
  protein--we began learning about this in
  Chp. 4
Eukaryotic RNA is processed before
       it leaves the nucleus

   Eukaryotic cells   Prokaryotic cells

                        RNA splicing …
                        (see next slide)
              RNA splicing
• Introns are
  removed, and
  exons are then
  spliced together
       Alternative RNA splicing
• When a single gene
  can result in different
  mRNA molecules b/c
  different combinations
  of exons are spliced
  together.                  RNA transcript


• Different mRNA
  molecules with different
  combinations of exons                        Alternative
                                              RNA splicing
  will encode different
   Alternative splicing increases the
    complexity of gene expression
• Single gene can
  be spliced into
  multiple mRNA
  leading to
  production of
  multiple proteins.
• In humans, each
  gene has an
  average of 3.5
  splice variants.
                       from Nature Genetics 36, 915 - 916 (200
      Overview of gene expression
        DNA--> RNA--> protein
• Can think about the process
  like a language:
  – DNA’s language: nucleic acid
                                        DNA “rewritten as RNA
                                        (in Chp. 5 notes)
  – RNA’s language: also nucleic acid
                                          Changing into the
  – Protein’s language: amino acid       language of protein

• Each 3 nucleotides of DNA (a triplet) is like a
  ―word‖ called a codon.
   – The three-nucleotide codon is translated into a single
     amino acid in the sequence of a polypeptide.
an overview

               Fig. 5.16
    3 kinds of RNA in the cell
• Messenger RNA (mRNA):
  – Encodes genetic information from DNA that
    will be translated into protein.
• Transfer RNA (tRNA):
  – Functions as an interpreter during translation.
  – Has an anticodon end that recognizes codons
    on mRNA and delivers the appropriate amino
    acid to a growing polypeptide chain.
• Ribosomal RNA (rRNA):
                                          Go back to
  – Makes up ribosomes, along with protein. 5.16 &
  – The most abundant form of RNA.        label all 3 …
an overview

               Fig. 5.16
 tRNA acts as an interpreter during
 translation of mRNA to polypeptide
                                           Amino acid attachment site

• tRNA’s are ~80 nucleotide single
  strands of RNA that fold & twist
  upon themselves.
                                                    Hydrogen bond
• On one end, they have a triplet of
  bases (called an anticodon) that is
  complementary to a codon in                    RNA polynucleotide
• On the other end, they bind to a
  specific amino acid (according to
  the anticodon)
                                                  Amino acid
• In this way, they deliver the correct         attachment site

  amino acid (specified by mRNA)(cartoon
  the growing polypeptide chain version)
                                                     True shape of a ribosome
Ribosomes                                       tRNA

• Ribosomes have 2 subunits                                                        Large
  – Both are made up of rRNA
    and protein.
  – The subunits hold the mRNA
    and tRNAs close together                     mRNA                          Small
    during translation.                                                        subunit

                                tRNA-binding sites   Cartoon of a ribosome
                                                                               Next amino
                                                                                acid to be
                                                                                added to

         mRNA-                                                                     tRNA
       binding site                                   mRNA
          subunit                                                     Codons
 Following transcription
 and translation of a
 small region of “gene 3”
                                                                   DNA molecule

                                                                                  Gene 1

• Transcription: DNA is rewritten as RNA.
                                                                                  Gene 2
   – Note that the RNA sequence is complementary
     to the DNA b/c the DNA is used as a template.
• Translation: RNA is changed into the                                   Gene 3

  language of polypeptides.
  (the primary structure of a single     DNA strand
  protein subunit).                                    A A A C C G G C A A A A

   – Each 3-nucleotide codon
                                                       U U U G G C C G U U U U
     instructs the addition of a            RNA

     specific amino acid into          Translation

     the polypeptide sequence.
   – Occurs in the cytoplasm.           Polypeptide
                                                      Amino acid
               The genetic code
• Is a set of rules that
  tell us how codons
  are translated into
  amino acids in
   – One codon can be
     Met or ―start.‖
   – 3 codons mean ―stop
   – Note the redundancy
     in the code.
      • But there is no
        ambiguity in the code
        (one codon can only
        specify one a.a.).
                                (Similar to Table 5.2 in your book)
An example: from DNA to polypeptide
                                  Strand to be transcribed

          T    A    C   T   T     C    A   A     A    A      T   C
          A    T    G   A   A     G    T    T    T    T   A      G


         A    U     G   A   A     G    U    U    U    U   A      G

           Start                                         Stop
          condon                                        condon

Polypeptide   Met           Lys            Phe
     Practice using the genetic code
                              Strand to be transcribed

          T   A   C   A   G    T   C    T   A     C   C   G
          A   T   G   T   C   A    G   A    T     G   G   C




                                                              Fill in the RNA
Polypeptide                                                   nucleotide bases and
                                                              these 4
                                                              amino acids
Q: Which of the following correctly ranks
   the structures below in order of size,
   from largest to smallest?

 a) gene-chromosome-nucleotide-codon.
 b) chromosome-gene-codon-nucleotide.
 c) nucleotide-chromosome-gene-codon.
 d) chromosome-nucleotide-gene-codon.
 e) gene-chromosome-codon-nucleotide.
              Initiation of translation
1) mRNA binds to the small ribosomal subunit and
  initiator tRNA binds to start codon (AUG).
     – Initiator carries the amino acid methionine (Met).
2) Large ribosomal subunit binds to the small one,
  creating a functional ribosome.
     – Initiator tRNA fits into the P site of the ribosome.
     – The other tRNA-binding site on the ribosome (the A site)
       is empty, awaiting the next amino-acid-bearing tRNA.
                        Met                                 Met
     Initiator tRNA
                                                 P site            A site
                      U A C                               U A C
                      A U G                               A U G
     mRNA                      Small ribosomal
 1                             subunit              2
          Translation elongation
1)   New tRNA carrying an                  Polypeptide
     amino acid binds to
                                                P site                   A site
     mRNA codon in the A                                                           Anticodon

     site of the ribosome.                  mRNA             Codons
                                                                        1   Codon recognition
2)   Polypeptide separates
     from the tRNA to which
     it was bound (the one in    mRNA
     the P site) and forms a
     peptide bond to the
     amino acid on the tRNA                              codon

     in the A site.                                                          2    Peptide bond
3)   The P site tRNA leaves                                           New
     the ribosome and the                                             bond

     ribosome translocates
     (moves) the tRNA in the
     A site (w/attached
     polypeptide) to the P                               3   Translocation
         Translation termination
• Elongation continues.
   – Ribosome moves down the mRNA one codon at a time, and tRNA’s
     complementary anticodon pairs with each codon, delivering its amino
     acid to the peptide chain until:
• A stop codon reaches the ribosome’s A site.
   – Stop codons (UAA, UAG, UGA) don’t code for amino acids but
     instead act as signals to stop translation.
   – The completed polypeptide is released from the last tRNA, and the
     ribosome subunits separate.

DNA--> RNA--> protein                      mRNA
                                                                                           1 mRNA is transcribed
                                                                                           from a DNA template.
                                           Amino acid                Translation
                                                                                            2 Each amino acid

• Transcription:                                                   Enzyme                  attaches to its proper
                                                                                           tRNA with the help of a
                                                                                           specific enzyme and ATP.

  – DNA--> RNA

  – In the nucleus.
                                            Initiator                       Large
                                            tRNA                            ribosomal      3 Initiation of
                                                                            subunit       polypeptide synthesis
                                                                                          The mRNA, the first tRNA,
                                                                                          and the ribosomal
                                                                                          subunits come together.
                                                           Start            Small

• Translation:                               mRNA
                                                           Codon            ribosomal

  – mRNA--> polypeptide
                                                                            New peptide
                                             Growing                        bond forming

                                                                                            4 Elongation

  – In the cytoplasm.                                                                      A succession of tRNAs
                                                                                           add their amino acids to
                                                                                           the polypeptide chain as
                                                                                           the mRNA is moved
                                                             Codons                        through the ribosome,
                                                                                           one codon at a time.

 (in prokaryotic cells, there is no need
 For RNA export from the nucleus to                                                        5 Termination
 the cytoplasm before translation)                                                       The ribosome recognizes
                                                                                         a stop codon. The poly-
                                                                                         peptide is terminated and
                                                             Stop codon                  released.
Q: An organism’s genetic information isThis
   within the sequence of ___________.

    information is transcribed into a sequence
    of ____________ which are then translated
    into a sequence of ___________.

      a) DNA bases, amino acids, RNA bases
      b) RNA bases, DNA bases, amino acids
      c) Amino acids, DNA bases, RNA bases
      d) DNA bases, RNA bases, amino acids
Different categories
  of proteins have
 different functions
 Functional categories of proteins
• Structural: (ex: collagen in skin, ligaments, tendons)
• Enzymes: increase rate (catalyze) chemical
  reactions in cells. (ex: amylase breaks starch down
  into sugar)
• Antibody: function in the immune system.
• Transport: ex: LDL carries cholesterol in the blood.
• Movement: proteins that contract or move (ex: actin,
  contractile protein important for muscle contraction.)
• Hormone: act as messengers between cells (ex:
• Pigment: absorb light, have color (ex: hemoglobin)
• Recognition: on cell surface; allows cells to
  recognize each other (ex: CD4 protein on T-cells)
  We’re going to learn about two of
   these categories in more detail

- Enzymes
- Antibodies
• These two categories of proteins are very
  relevant to the BT/BM field because many
  biomanufacturing products are
  – Enzymes, or regulators of enzymes
  – Antibodies, or vaccines designed to induce
    production of antibodies
Enzymes are protein catalysts
• Catalyst:
  – a chemical agent that speeds up a reaction
    without being consumed by the reaction
• Many biotech/biomanufacturing products
  are enzymes
  – Biomedical: many diseases involve an inability
    to produce a particular enzyme; patient
    replaces missing enzyme with bioengineered
    enzyme drug
  – Industrial: enzymes are required for many
    industrial applications and processes
  Reactions catalyzed by enzymes
• Enzymes are involved in virtually every reaction in a
• Some enzymes break molecules down into smaller
  – Ex: proteases break proteins down into amino acids
  – EX: sucrase breaks down sucrose into glucose + fructose
      sucrose                   glucose + fructose

• Some enzymes build larger molecules from smaller
  – EX: peptidyl transferase builds polypeptides by linking
    amino acids together
                                   DNA polymerase
  – EX: DNA polymerase builds DNA molecules from
Nucleotide1 + nucleotide2, etc.,
    nucleotides                                             DNA
        Enzyme nomenclature
      (how enzymes are named)
• Enzymes are often named for their
  substrates or for the function they perform,
  with the suffix ―-ase‖ added to the end of the
• Some examples:
- DNase breaks down DNA
- Proteases break down proteins
- Transferases transfer chemical groups
  between different molecules
            Enzyme terminology
• Substrates:
  – The molecules upon which enzymes act
  – Enzymes are usually very specific:
     • Each enzyme only has one or very few substrates
     • Each enzyme catalyzes only one type of chemical
• Active site:
  – pocket or groove on the enzyme surface that the
    substrate fits into--this is where the reaction occurs
• Induced fit:
  – When a substrate binds to an enzyme, the enzyme
    changes shape slightly so it can bind the substrate
    more tightly (kind of like clasping someone’s hand during a
                       Induced fit


Active site

              Enzyme            Enzyme bound to substrate
 •   This diagram shows the reaction catalyzed
     by the enzyme sucrase
     – What is/are the substrate(s) in this reaction?
     – What is/are the product(s) in this reaction?
    Four Features of Enzymes
1) Enzymes do not make anything happen that
   could not happen on its own. They just make
   it happen much faster.
2) Enzyme molecules are not changed during
   the reactions they catalyze.
3) Enzymes are very specific.
  - each type of enzyme binds only to certain
  - each type of enzyme catalyzes a specific reaction.
4) Enzymes are affected by the cellular
Each enzyme has its own optimal
   environmental conditions
• Temperature:
  – If too cold, substrate molecules move too slowly to
    contact enzyme active site
  – If too hot, the enzyme will denature (unfold)

                           Optimal temperature for   Optimal temperature for enzyme from
                           typical human enzyme      thermophilic (heat-tolerant) bacteria
    Rate of reaction

                       0           20          40        60          80         100

                                              Temperature (Cº)
Each enzyme has its own optimal
   environmental conditions
• pH:
       – Enzymes need the proper concentration of H+ ions to
         maintain protein structure & function.

                        Optimal pH for pepsin            Optimal pH
                        (stomach enzyme)                 for trypsin
 Rate of reaction


                    0     1      2       3      4   5      6      7    8   9   10

           Molecules that help
            enzymes function

• Cofactors
  – Are nonprotein enzyme helpers
  – Can be inorganic (e.g. zinc, iron, or copper ions)
  – Can be organic molecules, in which case they
    are called coenzymes.
 Poisons, pesticides, & drugs are
     often enzyme inhibitors
• Cyanide: inhibits an enzyme that produces ATP
  (cellular energy storage molecule)
• Sarin (a nerve gas): inhibits acetylcholinesterase, an
  enzyme essential for nerve cell function—causes
  paralysis & death
• Pesticides malathion & parathion: also inhibit
  acetylcholinesterase, but in insects
   – Can also be toxic to other animals, including humans
• Many antibiotics inhibit enzymes in bacteria.
• Aspirin, ibuprofen: inhibit enzymes that induce
  inflammation & pain.
• Protease inhibitors treat HIV by inhibiting a viral
• Function: to recognize and bind foreign
  molecules (called antigens), ultimately to
  remove the antigens from the body.
• Antibodies recognize antigens with great
  specificity --recognition works like a lock and
  key                       Antibody A
                           molecules          Antigen-
  – Antigens may be proteins,                 binding
    carbohydrates, or other
  – Different antibodies
    can recognize different         en
    parts of an antigen           molecule
                                Antibody B
 How antibodies are made in the body
• Antigen enters the body                   Different B cells
  and encounters B cells (a
  type of white blood cell).
• As a result of random           Antigen binds
  reshuffling in the DNA of       to B cell that
                                  has antibody on
  antibody genes, every B         its surface that
  cell expresses and              fits the antigen
  ―presents‖ a different
  antibody molecule on its
  cell surface
• If this antibody binds to its
  antigen (lock and key),
  then that B cell is
                                    B cell is stimulated to make more
  stimulated to reproduce,          of the same antibody and secrete it
   Structure of antibody molecules
• Each antibody molecule is made up       Antigen-
  of 4 polypeptide chains, with the        sites

  same overall shape
• 2 identical ―heavy‖ chains and 2                         Light
  identical ―light‖ chains                                 chain

• The variability in antibody               C    C Heavy
  molecules is found at the top of                 chain
  the ―Y‖, in the variable regions (see
                                             (similar to Fig. 5.8)
  V’s in diagram) --this region
  forms the antigen-binding site.
• The tail of the antibody has
  constant regions (see C’s in
  diagram) that allow the antibody to
  interact with other components of                   (similar to
                                                       Fig. 5.9)
  the immune system
          Antibodies in BT/BM

 Because they recognize & bind to molecules
 with such specificity, antibodies are very
 useful in:
 Purification of proteins from cell cultures
 (more on this in Chp. 9)
 Diagnostic tests
 Biopharmaceutical therapies
 Research tools to visualize proteins in cells
Antibodies as tools for purification
• Purification technique called affinity purification
  (more on this in Chp. 9)
• Allows a specific protein to be purified from cell lysate
  (a mixture of cellular
  molecules)             Protein
• Purified proteins are by antibody
  common products        Protein not   Wash    Elute
  in biomanufacturing recognized
                         by antibody

      Antibodies in diagnostic tests
• Antibodies can detect the presence of molecules that
  indicate a specific medical condition:
• Some examples:                     Staining breast biopsies for
                                     breast cancer markers
   – Pregnancy tests
   – Tumor markers in biopsies
   – HIV antibody tests
     (see next slide)

                    Pregnancy test
                    using antibodies
                    against a
                    hormone, hCG

                                          Götte et al. Breast Cancer Research 2007
                           HIV antibody test

If a person is HIV+,          Blood is added to a dish     If HIV antibodies are
their blood will contain      containing many different    present in the blood, they
antibodies against HIV        HIV virus antigens           will bind to the HIV antigens

                                                                                  yellow =

           A chemical is added that turns    Results look like this; a positive
           yellow if it recognizes any HIV   result will be rechecked by an
           antigen-antibody complexes        alternate method
 Antibodies as biopharmaceuticals
• Once an antigen has been identified as a disease
  target, antibodies can be made that bind to and
  possibly inactivate the antigen, reversing or
  lessening symptoms
• EX: Anticancer therapeutic antibodies:
  – may mark the cancer cell for immune system
  – Can block binding sites for growth factors on cancer
  – May be attached to a chemotherapy drug that kills
    cancer cells
• EX: anti-TNF targets protein involved in
 Antibodies as biopharmaceuticals

Some examples of antibodies that have been
approved as cancer treatments
 Antibodies as research tools for
         visualizing proteins in cells
• Knowing the location of a protein or other
  molecule inside a cell can reveal important
  information about its function and role in normal
  and disease physiology
• Antibodies are used to ―stain‖ cells; then, the
  antibodies can be detected using a fluorescent or
                          Immunofluorescence microscopy using an
                         antibody against a protein involved in cancer
  colored marker
  to see where the
  antigen is located
  inside the cell

                                normal mammary cells   breast cancer cells
     How antibodies are made, part 1
• Antigen X is injected into an animal (usually a
  mouse, rabbit, sheep, or goat)
   – Usually, injections of the same antigen are repeated at
     intervals of several weeks
• Antigen X injections stimulate specific B cells to
  secrete large amounts of anti-X antibodies into the
• Later, blood is taken from the animal, and the anti-X
  antibodies are purified
• Because many different B cells
  will be stimulated by                 X
  antigen X, a variety of
  anti-X antibodies
  will be produced;
  each one binds
  antigen X in a slightly
                        inject antigen X     take blood later
  different way
  How antibodies are made, continued
• In the previous slide, all the anti-X antibodies will
  recognize and bind to antigen X, but they will all
  be different and therefore bind to antigen X in
  slightly different ways
  – They are all different because they came from different
    B cell populations that recognized antigen X
  – The anti-X antibodies produced from the method in the
    previous slide are called polyclonal antibodies
• Sometimes it is necessary to have all the anti-X
  antibodies be identical and recognize antigen X in
  the exact same way
  – This is especially important for antibodies that will be
    used as therapeutic drugs
  – For this, it is necessary to create a monoclonal
     How antibodies are made, part 2
     Making monoclonal antibodies
• Mouse is injected w/antigen X;
  B cells make anti-X antibodies Antigen injected              Tumor cells
• Anti-X-producing B cells are into mouse                    grown in culture

  isolated and fused with tumor
  cells to produce hybrid cells B cells (from spleen)          Tumor cells
   – Tumor cells are used to
     create hybrids that will are easy
     to grow and will divide indefinitely
                                                                   Cells fused
• Single hybrid cell is selected and                               to generate
                                                                   hybrid cells
  grown in culture
                                                            Single hybrid
   – Since it’s from only one hybrid, only one              cell grown in
     type of anti-X antibody will be produced

                                            Hybrid cell culture, producing
                                               monoclonal antibodies
            Studying proteins:
Properties of proteins that researchers need
  to know when studying a specific protein
 1) Amino acid sequence (primary structure)
 2) Molecular weight
   – can be roughly estimated from the # of amino
   – can be determined exactly given the amino acid
 3) Net charge
   – Since some amino acids are charged, proteins
     have a characteristic overall ―net charge‖
     depending on which amino acids make up the
           Studying proteins
• There are many sophisticated techniques for
  studying protein characteristics . . . We’ll
  discuss more of these in Chp. 14
• In this chapter, we will discuss studying
  proteins by gel electrophoresis
Review from Chp. 4:
• Gel electrophoresis: laboratory
  technique that uses electricity and a thin
  gel to separate molecules (usually, nucleic
  acids or proteins)
• Molecules are separated by size, shape,
Polyacrylamide gel electrophoresis
 • Like agarose gels, polyacrylamide gels are
   prepared as a liquid, and allowed to solidify
   – Note: liquid polyacrylamide is highly toxic (safe
     once it has polymerized)
 • Polyacrylamide gel electrophoresis (PAGE)
   is used for smaller molecules (nucleic acids
   <500 bp, proteins)
   – The pores in polyacrylamide gels are smaller
     than agarose gels, so polyacrylamide gels are
     better able to resolve (separate clearly) smaller
        PAGE uses vertical gel
• Liquid polyacrylamide is mixed with buffer
  and poured between two vertical glass
• A comb is inserted between the plates to
  create the wells into which the protein
  sample will be loaded
• Stands for ―SDS Polyacrylamide Gel
• Similar in many ways to agarose gel
  electrophoresis that is used to separate DNA
    1) Thin gel separates molecules on the basis of size.
    2) Negatively charged molecules are loaded into wells
       a gel that is submerged in buffer.
    3) Electrical current is applied to the gel, causing the
       molecules to move toward the positive electrode
       anode, usually marked red)
    4) Smaller molecules move faster through the gel than
• Some differences from agarose gel
   1) SDS-PAGE is usually used to separate protein
       molecules; agarose gels are usually used to
       separate DNA molecules.
   2) Proteins must be coated with SDS before
      being loaded in the gel
    - SDS is a negatively charged detergent
      that gives the proteins a negative charge
      and denatures them (makes them linear
      instead of 3-D).
     (note: DNA is already negatively charged and linear, so don’t need
   Purpose of adding SDS in SDS-PAGE:
        SDS serves two functions

 Coats proteins
 with negative
so they will move
toward the positive

 Makes protein
so they migrate
according to their
molecular weight in
          SDS-PAGE, cont.
 Samples are  Electrical    Molecules
 loaded into   current is    are
 wells         applied         separated by
                               size largest

How Does an SDS-PAGE Gel Work?
• Negatively charged
  proteins move to           s-s

  positive electrode               Proteins with
                                   SDS & heat
• Smaller proteins       –
  move faster
• Proteins separate
  by size                +
  – Largest at top
  – Smallest at bottom
 After you’ve run the SDS-PAGE gel,
    how do you see the proteins?
1) Stain the gel
  – Many different
    stains out there,
    with varying
2) Western blot
  – Allows you to identify a particular protein of
    interest among many proteins in a sample by
    using an antibody that recognizes your protein
    of interest
    Coomassie                        Silver stain
    blue stain
                              • Stains all proteins
• Stains all proteins present   present
• Limit of detection:         • Much more sensitive
  – In theory: 50-100 ng              – But also more expensive
  – In practice: need at least 0.5      and time-consuming
    µg                             • Limit of detection: 1-10
                  Protein Size
• Size measured in kilodaltons (kD)
  – (D stands for dalton, a unit used for molecular
    weight); 1 dalton = 1 g/mol
  – Average amino acid = 110 daltons
  – Most proteins are between 10 - 150 kD

• Molecular weight markers are always run
  next to samples on SDS-PAGE gels
  – MW markers are commercially available
    mixtures of proteins of known molecular weight.
  – The marker is necessary to compare with the
    sizes of the proteins in the samples (see picture,
    next slide)
      Example of an SDS-PAGE gel

• Q: What is the
  approximate MW
  of the protein in
  sample #2?
  What about
  sample #1?
   Gels of different concentrations
• Smaller molecules are run on higher concentration
• Larger molecules are run on lower concentration gels
• Gels can also be made with a concentration gradient
  (more concentrated at the bottom of the gel than the
   removed or
extraneous slides
Everything that’s needed for

     1)   mRNA molecules
     2)   tRNA molecules
     3)   Amino acids
     4)   Ribosomes
     5)   ATP for energy
       A start codon marks the
    beginning of an mRNA message

Start of genetic message   • mRNA sequences before start
                             codon and after stop codon do
                             not get translated.
                                - These extra sequences help
                                  the mRNA bind to the
                                  ribosome during translation.


 Figure 10.13A
Antibodies mark antigens for elimination
 • Using several different effector mechanisms:
    – Note that specific recognition phase is followed by a
      nonspecific destruction phase.
                                              Binding of antibodies to
                                          antigens inactivates antigens by

                Neutralization        Agglutination
                                       (clumping)              Precipitation of         Activation of
            (blocks viral binding
 specific   sites; coats bacteria)     of microbes           dissolved antigens      Complement system

acquired                                                                                            Complement
 immune                 Virus                   Bacteria                                             molecule

            Bacterium                                                    molecules   Foreign cell         Hole

                                       Enhances                                           Leads to

  nonspecific                         Phagocytosis                                       Cell Iysis
                        Figure 24.9           Macrophage
                      Enzyme Inhibitors
              A substrate can
          bind normally to the                              Active site
              active site of an

                                  Normal binding
                                                                              A noncompetitive
                                                                              inhibitor binds to
                                                                              the enzyme away
   A competitive                                                              from the active site,
inhibitor mimics                                                              altering the shape
   the substrate,                  Competitive                                of the enzyme so
   competing for                                                              that its active site
  the active site.                                                            no longer binds
                                                   Noncompetitive inhibitor   substrate.

Competitive inhibition                           Noncompetitive inhibition
What’s in • Tris buffer to provide
  the       appropriate pH
Sample • SDS (sodium dodecyl
Buffer?     sulfate) detergent to
              denature proteins and give
              them a negative charge
             • Glycerol to make samples
               sink into wells
             • Blue dye to visualize
               samples as gel is run

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