lab 7 SDS by 9WvuL0lX

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									                The usual
• Pre and post lab exercises are on handouts
• Online quiz
• Don’t return today or tomorrow
  SDS-PAGE OF myoblasts, myotubes,
    fibroblasts and adipocyte lysates

• Sodium Dodecyl Sulfate Polyacrylamide Gel
  Electrophoresis
• Is a technique allowing for the separation of
  proteins based on their size.
• We will be loading
  –   Cell lysates of fibroblasts
  –   Cell lysates of adipocytes
  –   Cell lysates of myoblasts
  –   Cell lysates of myotubes
  –   Standard of proteins with known sizes
               Sodium Dodecyl Sulfate
• A negatively charged detergent
  which binds to hydrophobic regions
  of protein molecules
• Causes the molecules to unfold
   – from a globular into
      extended/linear polypeptide
      chain
• Individual protein molecules are
  released from their associations with
  other proteins or lipid molecules by
  SDS and are rendered soluble in the
  detergent solution.
• All proteins treated with SDS will
  be carry a net “negative” charge
              Polyacrylamide Gel
• Gels are formed by polymerization of acrylamide
  and bisacrylamide (ie. the polyacrylamide).
   – Small proteins run quickly through meshwork of
     polyacrylamide.
   – Larger, linear proteins get hung up in the gel and migrate
     slower
• Ammonium persulfate initiates the reaction.
• TEMED catalyzes the polymerization.
• Unpolymerized acrylamide is a potent neurotoxin.
Electrophoresis
• Process of migration of
  charged molecules through
  solutions in an applied
  electric field
• Negative charge at the top
  of the gel, Positive at
  bottom
• SDS treated proteins will
                 negative
  have a net ____________
  charge and will migrate
  toward __________ pole
              positive
 PAGE Gels Run Vertically
...unlike DNA agarose gels that run horizontally

         Loading Well

                            Cathode (- pole)

         Stacking Gel


                             Direction of
                          Protein Migration
         Separating Gel




                            Anode (+ pole)
   To clear up the anode/cathode
             confusion:
• Cathode: The electrode at which reduction occurs.
  It is the negative electrode in a cell through which
  current is being forced, but it is the positive pole of a
  battery.
• Anode: The electrode at which oxidation occurs in
  a cell. It is the electrode toward which anions travel
  due to the electrical potential, In spontaneous cells
  the anode is considered negative. In
  nonspontaneous or electrolytic cells the anode is
  considered positive.
• From the CRC Handbook of Physics and Chemistry
                       Stacking Gel
• Low percentage of                   Proteins should have
  acrylamide (2.5%-4%)                same starting reference
   – large pore size                  point when they enter
   – does not restrict migration of   the separating gel.
     proteins.                           It’s like getting all
• Ion contrast between those             the horses lined up
  that compose the buffer of             at the gate...
  the stacking gel to those in
  the electrode buffer allows
  the proteins to move quickly
  through this layer and allows
  them to “stack”.
                  Separating Gel
• Higher percentage (7.5%-20%) of acrylamide
• Pore size is smaller than the stacking gel
• Proteins separated logarithmically by their size
  (molecular weight).
   – Large proteins have difficult time maneuvering
     through the gel and move a shorter distance than
     smaller proteins that move easily and rapidly.
                   2.5
                   %

Protein SO-250kD



                   15
                   %



Protein JS-55kD
          Adjusting pore size for protein resolution
• Use different concentrations of acrylamide in order to study different size proteins
   – higher percentage will separate smaller sized proteins better.
   – A gradient will space the proteins evenly (ex 4-20%)
• This week we will use a 10-20% gel
         Mobility of a molecule
Mobility of a molecule =
(Applied voltage)(net charge on the molecule)
       (friction of the molecule)
       OR
       V = Ez
              f
       V =velocity (rate of migration)
       E=strength of electrical field
       z=charge on the molecule
       f=frictional force on the molecule
                   Other Reagents
b-mercaptoethanol                Glycerol
                                 • Increases the density of the
• Reduces disulfide bonds          sample so it will settle to the
  which cause proteins to          bottom of the well during
  fold or two protein subunits     loading
  to be bound together.
• Added to the sample to be      Bromophenol Blue
  run on the gel in order to     • A small dye added to the
                                   samples. The dye runs
  linearize and separate           faster than the protein
  proteins and their subunits      samples and gives us a dye
• Smells bad!!                     front so we can visualize
                                   the migration of the proteins
                                   (we won’t be able to see the
                                   proteins)
       Electrode Buffer
• Contains salts, electrolytes and
  buffers which are necessary for
  maintaining proper pH and
  conducting a current.
         Molecular Weight Markers
• A combination of proteins of
  known molecular weights in
  one solution.
• Used to create a standard
  graph of the distance proteins
  migrate and molecular size
  (on log paper)
• Can measure the distance of
  an unknown protein migrated
  and correlate its molecular
  weight in daltons or
  kilodaltons
                      Transfer dye/ladders
• Transfer dye
  – A dye remains with the proteins to help
    visualize transfer of proteins onto the
    nitrocellulose
• Ladder
  – Most molecular weight standards are
    blended from naturally occurring proteins
     • Inherent variability in the amount and location
       of dye that covalently binds to the protein
         – Produces diffuse, broader bands
  – Carefully engineered for precise and
    accurate molecular weights from lot to lot
   The standard
      curve
• Using log paper or a
  website/program
  designed for standard
  curve creations, plot the




                              Molecular weight
  known molecular
  weights of the standard
  proteins against their
  distances migrated
• Then, find some strong
  bands in your lysate run
  and measure those.
  Calculate their
  approximate weights.
                                                 Distance migrated in mm
            Skeletal muscle lysates
Myoblasts
• Single cell precursors to muscle
• Do not produce many proteins seen in mature muscle like
  myosin heavy chain (MHC) mw: 200kD

Myotubes
• Are created by the fusion of myoblasts to form long muscle
  cells with several nuclei (syncitium)
• Produce MHC and actin
   – forms contractile machinery
myoD1
   – The gene responsible for differentiation of a myoblast
      into a mature skeletal muscle cell
         Fibroblast and adipocytes
• Adipocytes and Adipsin
• For 3T3-L1s to differentiate to adipocytes, we add
  insulin, dexamethasone and IBMX.
   – There is a transient increase of DNA synthesis 18h
     after adding IBMX followed by an arrest in the G1
     cycle (stop growing temporarily)
   – During this arrested time, a few genes are turned on
     that lead to differentiation.
                    Adipsin
• One gene turned on is the one that produces
  adipsin
• Adipsin
   – a serine protease
      • Oddly enough, PMSF is a serine protease
        inhibitor
   – Secreted only by adipocytes in most animal
     models (in humans is also expressed in
     monocytes/macrophages)
   – Is deficient in several animal models of obesity
   – Has been identified as the same protein as
     complement factor D
   – Is about 22kD
            FYI: Complement cascade
• About 20 normal serum
  proteins
   – Ranging from 24kD-550kD
• Work with antigen bound
  antibodies                              Adipsin, (complement D
                                          or C3 convertase activator) is
• Various biological effects              About step 6 or 7 of 9 steps before
                                          opsonization
   – Lyse invading cells
   – Initiates inflammatory response
   – Opsonization
      • Coating on surface of substance
         – Enhances phagocytic activity
            by macrophages and
            neutrophils (white blood
            cells)
     What Can You Do With A Gel?
Stain it                            Western Blot
• Fix proteins into gel by          • Transfer proteins to a
  immersing in methanol and           substrate that’s easier to
  acetic acid                         handle and “blot” or
• Stain gel with “Fast Stain.”        “probe” with specific
                                      antibodies. (next week)
   – modified Coomasie blue,
     allows for shortened destain   • FYI:
     procedure.                     • Northern blot
• Destain in 10% acetic acid.          – DNA
• Photograph gel.                   • Southern blot
• Calculate standard curve and         – RNA
  MW of observed proteins.
• Generally, the lanes of lysate will
  look like big smears punctuated by
                                            Staining
  heavier bands after staining the gel           MW Mt         Mb   adp
  because there are a lot of proteins of
  various sizes. Stain is for total      200 kDa

  protein.
                                                     166 kDa
• Myoblast and myotube Lanes                         97 kDa
   – Expect to see a band for myosin around 200
     kDa in the myotube lane but not the myoblast    66 kDa
     lane
   – Generally more proteins in the myotube lane.
   – If we see a band near the myosin range in the
     myoblast area, a couple of reasons could be     45 kDa
       • The myoblasts already began to
         differentiate
       • Sample contamination
            Example of a “good” gel
• Can see “smears” of
  proteins
   – Proteins come in the full
     spectrum of sizes
• There are distinct bands
  embedded within
   – Representing large quantities
     of intact proteins
   – Lack of distinct bands
     represents poor lysate          Curving of bands in side wells
     technique where the             is a fairly common artifact
     proteases began chewing up
     the proteins                    Pale lanes for undifferentiated
                                      cell lysates is to be expected
                      The Western Blot
• Transferring proteins from a gel      • Nitrocellulose is placed
  to a nitrocellulose membrane            against the gel and a current
  in order to probe for a specific        is used to pass the proteins
  protein                                 from the gel to the membrane
   – use antibodies against a epitope     where they will bind
     specific to the target protein.
                                           – again, proteins will run toward
• Nitrocellulose is used because it          the positive pole
  is easier to work with a
  membrane than a gel
• Nitrocellulose has a high
  affinity for proteins, even the
  ones on your fingers, so wear
  gloves!!
• Will use blotting buffer
   – different balance of salts,
     electrolytes, etc. than the gel
     running (electrode) buffer        • Use an ice block to keep system
   – need to soak gel in new             cool (high currents) so gel
     buffer or gel will shrink           won’t melt and proteins won’t
     during blotting                     smear
   – has methanol that removes         • If a transfer dye was not
     SDS from proteins, allowing
     some re-naturation and              utilized, could use a stain called
     better antibody recognition         Ponceau S.
   – keeps proteins from                  – This would allow total protein
     diffusing away                         visualization
   – enhances protein binding to
     membrane and helps gel
     maintain size and shape
     during blotting
          Media, an incomplete list
• MEM
  – Minimum Essential Media
  – Sometimes called Eagle’s MEM or
    Basal Medium Eagle (BME)              • Ham’s F12 (Nutrient
                                            Mixtures)
  – First widely used media, supports a
    broad spectrum of mammalian cells       – Complex formula
                                               suitable for serum
  – Developed by Harry Eagle (1950s)           free propagation
• DMEM                                      – DMEM and F12
  – Dulbecco’s Modified Eagle Media            can be mixed
  – 2x the [amino acids] and 4x the            together to support
    vitamins of MEM                            a broad range of
                                               cells
  – Most often contains 4.5mg/L glucose
                                            – First developed to
  – Standard medium for mammalian
                                               support CHO cells
    cells
                          More Media
                                         • L15 Leibovitz
• RPMI and McCoy’s                         – Can be used without CO2
  – Developed at Roswell Park                incubation (caps of flasks can
    Memorial Institute in Buffalo            stay tightly closed!)
    New York.                              – Found in teaching laboratories
  – McCoy’s was originally used to           or when collecting biopsy
    grow hepatoma cells and                  samples
    supports growth of primary             – Standard sodium
    cultures                                 bicarbonate/CO2 buffering
  – RPMI is modified McCoy’s                 system is replaced by
     • Long term culture of peripheral         • Combo of phosphate buffers
       blood lymphocytes                         (Hank’s balanced salt
     • Suspension or monolayers                  solution)
     • Suitable for a wide variety of          • Free-base amino acids
       suspension cultures                     • Higher levels of sodium
                                                 pyruvate and galactose
             Even more media
                            • Iscove’s modified
• Glasgow minimum             Dulbecco’s media
  essential media (G-         – Suitable for fast
  MEM)                          growing, high density
  – Developed for the           cell cultures
    culture of baby           – Highly enriched
    hamster kidney cells        synthetic media
    (BHK-21)                • Medium 199
• Neurobasal media            – Popular for fibroblast
  – Specially designed to       cultures
    support neurons           – Originally formulated
                                for chick embryo
                                fibroblasts
              Balanced Salt Solutions
• Inorganic salts and may          • PBS
  include sodium bicarbonate       • Earle’s buffered salts
  and sometimes glucose              solution (EBSS)
   – Diluent for amino acid           – Higher bicarbonate
     concentrates and vitamins          concentration compatible
   – Isotonic wash or dissection        with growth in 5% CO2
     medium
                                   • Hank’s buffered salts
                                     solution (HBSS)
                                      – Good for sealed flasks
                                        with a gas phase of air
    Media Buffers, a short list
• Vary in their pKa values and toxicity to cells
• Sodium bicarbonate
   – Most common and most liquid medias are prepared
     with this.
• HEPES
   – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
   – Can be toxic for differentiated cell types
   – Can increase sensitivity of media to phototoxic effects
     induced by exposure to fluorescent light
• Bufferall
   – Contains 3 biological buffers
   – Very good a reducing pH fluctuations

								
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