The usual

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The usual

• Pre and post lab exercises are on handouts
• Online quiz
• Dont return today or tomorrow

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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

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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

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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.

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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 have a net ____________
  charge and will migrate toward __________ pole

negative
positive
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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)
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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

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Stacking Gel
Proteins should have same starting reference
point when they enter the separating gel. Its
like getting all the horses lined up at the
gate...

• Low percentage of acrylamide (2.5-4)
• large pore size
• does not restrict migration of proteins.
• Ion contrast between those that compose the
  buffer of the stacking gel to those in the
  electrode buffer allows the proteins to move
  quickly through this layer and allows them to
  stack.

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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.

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Protein SO-250kD
Protein JS-55kD
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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

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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)
• Estrength of electrical field
• zcharge on the molecule
• ffrictional force on the molecule

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Other Reagents

• Glycerol
• Increases the density of the sample so it will
  settle to the bottom of the well during loading
• Bromophenol Blue
• A small dye added to the samples. The dye runs
  faster than the protein samples and gives us a
  dye front so we can visualize the migration of
  the proteins (we wont be able to see the
  proteins)

• b-mercaptoethanol
• Reduces disulfide bonds which cause proteins to
  fold or two protein subunits to be bound
  together.
• Added to the sample to be run on the gel in order
  to linearize and separate proteins and their
  subunits
• Smells bad!!

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Electrode Buffer

• Contains salts, electrolytes and buffers which
  are necessary for maintaining proper pH and
  conducting a current.

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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

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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

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The standard curve

• Using log paper or a website/program designed for
  standard curve creations, plot the 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.

Molecular weight
Distance migrated in mm
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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

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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.

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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

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FYI Complement cascade

• About 20 normal serum proteins
• Ranging from 24kD-550kD
• Work with antigen bound antibodies
• Various biological effects
• Lyse invading cells
• Initiates inflammatory response
• Opsonization
• Coating on surface of substance
• Enhances phagocytic activity by macrophages and
  neutrophils (white blood cells)

Adipsin, (complement D or C3 convertase
activator) is About step 6 or 7 of 9 steps
before opsonization
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What Can You Do With A Gel?

• Western Blot
• Transfer proteins to a substrate thats easier to
  handle and blot or probe with specific
  antibodies. (next week)
• FYI
• Northern blot
• DNA
• Southern blot
• RNA

• Stain it
• Fix proteins into gel by immersing in methanol
  and acetic acid
• Stain gel with Fast Stain.
• modified Coomasie blue, allows for shortened
  destain procedure.
• Destain in 10 acetic acid.
• Photograph gel.
• Calculate standard curve and MW of observed
  proteins.

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• Generally, the lanes of lysate will look like big
  smears punctuated by heavier bands after staining
  the gel because there are a lot of proteins of
  various sizes. Stain is for total protein.
• Myoblast and myotube Lanes
• Expect to see a band for myosin around 200 kDa in
  the myotube lane but not the myoblast 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
• The myoblasts already began to differentiate
• Sample contamination

Staining
MW
Mt
Mb
adp
200 kDa
166 kDa
97 kDa
66 kDa
45 kDa
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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
  technique where the proteases began chewing up
  the proteins

Curving of bands in side wells is a fairly
common artifact
Pale lanes for undifferentiated cell lysates is
to be expected
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The Western Blot

• Transferring proteins from a gel to a
  nitrocellulose membrane in order to probe for a
  specific protein
• use antibodies against a epitope specific to the
  target protein.
• Nitrocellulose is used because it 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!!

• Nitrocellulose is placed against the gel and a
  current is used to pass the proteins from the gel
  to the membrane where they will bind
• again, proteins will run toward the positive pole

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• Will use blotting buffer
• different balance of salts, electrolytes, etc.
  than the gel running (electrode) buffer
• need to soak gel in new buffer or gel will shrink
  during blotting
• has methanol that removes SDS from proteins,
  allowing some re-naturation and better antibody
  recognition
• keeps proteins from diffusing away
• enhances protein binding to membrane and helps
  gel maintain size and shape during blotting

• Use an ice block to keep system cool (high
  currents) so gel wont melt and proteins wont
  smear
• If a transfer dye was not utilized, could use a
  stain called Ponceau S.
• This would allow total protein visualization

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Media, an incomplete list

• MEM
• Minimum Essential Media
• Sometimes called Eagles MEM or Basal Medium
  Eagle (BME)
• First widely used media, supports a broad
  spectrum of mammalian cells
• Developed by Harry Eagle (1950s)
• DMEM
• Dulbeccos Modified Eagle Media
• 2x the amino acids and 4x the vitamins of MEM
• Most often contains 4.5mg/L glucose
• Standard medium for mammalian cells

• Hams F12 (Nutrient Mixtures)
• Complex formula suitable for serum free
  propagation
• DMEM and F12 can be mixed together to support a
  broad range of cells
• First developed to support CHO cells

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More Media

• L15 Leibovitz
• Can be used without CO2 incubation (caps of
  flasks can stay tightly closed!)
• Found in teaching laboratories or when collecting
  biopsy samples
• Standard sodium bicarbonate/CO2 buffering system
  is replaced by
• Combo of phosphate buffers (Hanks balanced salt
  solution)
• Free-base amino acids
• Higher levels of sodium pyruvate and galactose

• RPMI and McCoys
• Developed at Roswell Park Memorial Institute in
  Buffalo New York.
• McCoys was originally used to grow hepatoma
  cells and supports growth of primary cultures
• RPMI is modified McCoys
• Long term culture of peripheral blood lymphocytes
• Suspension or monolayers
• Suitable for a wide variety of suspension
  cultures

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Even more media

• Iscoves modified Dulbeccos media
• Suitable for fast growing, high density cell
  cultures
• Highly enriched synthetic media
• Medium 199
• Popular for fibroblast cultures
• Originally formulated for chick embryo fibroblasts

• Glasgow minimum essential media (G-MEM)
• Developed for the culture of baby hamster kidney
  cells (BHK-21)
• Neurobasal media
• Specially designed to support neurons

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Balanced Salt Solutions

• PBS
• Earles buffered salts solution (EBSS)
• Higher bicarbonate concentration compatible with
  growth in 5 CO2
• Hanks buffered salts solution (HBSS)
• Good for sealed flasks with a gas phase of air

• Inorganic salts and may include sodium
  bicarbonate and sometimes glucose
• Diluent for amino acid concentrates and vitamins
• Isotonic wash or dissection medium

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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