IGP Methodology: Western Blotting and ELISA

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IGP Methodology Western Blotting (and ELISA)

• A.J. Robison
• Colbran and Winder Labs
• Dept. of Molecular Physiology Biophysics
• Oct. 27, 2005
• a.j.robison_at_vanderbilt.edu

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Western blotting

• Purpose Detect specific antigens (proteins)
  recognized by polyclonal or monoclonal
  antibodies
• -Used after proteins separated on SDS-PAGE gel
• Uses
• 1. Qualitative
• Determine if a protein is present in a certain
  cell type/tissue
• Confirm protein expression in transformed or
  transfected cells
• 2. Quantitative
• Detect levels of a protein over time
• Detect protein levels in response to specific
  treatment

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SDS-PAGE

• SDS Sodium Dodecyl Sulfate
• PAGE Polyacrylamide Gel Electrophoresis

• Separation of proteins, based on size and charge,
  as they move through polyacrylamide gel matrix
• Sample Preparation
• Boil sample in sample buffer prior to loading
• Sample buffer
• contains SDS, dye, and 2-Mercaptoethanol
• (reduces disulfide bonds)

Coomassie stained gel
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SDS-PAGE

• Separation of proteins, based on size as they
  move through polyacrylamide gel matrix
• of polyacrylamide in gel alters saparation

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Basic Steps of Western Blot

• Transfer gel to membrane
• ?
• Block
• ?
• Primary antibody
• ?
• wash
• ?
• Secondary antibody
• ?
• wash
• ?
• Detection

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ELISA Basic Steps

• Coat well with antibody
• ?
• wash
• ?
• Add antigen
• ?
• wash
• ?
• Enzyme-conjugated antibody
• ?
• wash
• ?
• Detection with substrate

Enzyme-Linked Immunosorbent Assay
sandwich
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Transfer

• Transfer proteins separated by SDS-PAGE onto
  membrane
• membrane Nitrocellulose, polyvinylidene fluoride
  (PVDF)
• Purpose
• covalently attach proteins to membrane
• Membrane easier to handle than gel
• Proteins bound to surface of membrane, readily
  accessible to antibodies

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Tank Transfer - advantages

• More efficient transfer of high molecular weight
  proteins (gt100kDa)
• Transfer overnight
• Can transfer multiple gels at same time with
  equal efficiency
• Can reuse some buffers in rig multiple times

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Semidry Transfer - advantages

• 1. Uses less buffer (100 mL or less)
• -Can transfer multiple gels at same time, though
  with loss of efficiency
• 2. Takes less time
• (10-30 minutes
• vs. gt1 hour for tank)
• Tip different methods of transfer have
  different efficiencies - make sure to confirm
  efficient transfer when using a new method for
  the first time

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Protein Visualization

• Purpose Stain proteins on membrane to detect
  transfer efficiency, equal protein loading
• Can scan and use to normalize signal obtained by
  western blot (good for accurate quantitation)
• Fast Green, India ink, Ponceau S

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Blocking

• Purpose
• Fill any available protein-binding sites on blot
  with nonreactive protein to prevent non-specific
  antibody binding
• Most common blocking agents
• nonfat dry milk, casein, bovine serum albumin,
  serum (goat)

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Primary Antibody

• Purpose Use antibody that specifically
  recognizes and binds to protein of interest
• monoclonal (mouse) or polyclonal (rabbit, goat,
  sheep, etc.)
• Tip use positive and negative controls

Controls positive 1. purified protein of
interest 2. tissue known to contain protein
of interest negative 1. preincubate primary
antibody with antigen 2. tissue known to not
contain protein of interest
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First Wash

• Purpose Thoroughly wash away any unbound primary
  antibody
• Perform 5 5-minute washes using TTBS with
  constant agitation on rocker or shaker

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Secondary Antibody

• Purpose
• (a) Binds to the primary antibody
• (b) Conjugated to enzyme (etc.) that provides
  means of detection
• Species specificity is important
• Must be specific for the IgG of the species from
  which your primary antibody was generated
• Ex goat anti-mouse, rabbit anti-goat, sheep
  anti-rabbit
• If primary antibody is goat anti-PoI, use an
  anti-goat secondary

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2 Antibody Conjugates

• Enzymes chromogenic, chemiluminescent
• Alkaline Phosphatase
• -Sharp bands
• -Control exposure in real time
• -cant strip membrane
• HRP/ECL
• -bands not as sharp
• -can strip/reprobe
• -make multiple exposures
• Infrared dyes
• -Expensive
• -can strip/reprobe
• -sharper bands than ECL
• Other Radiochemicals (125I-protein A),
  Biotin/avidin, Colloidal gold,
  Fluorescent compounds

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Second Wash

• Purpose
• Thoroughly wash away any unbound secondary
  antibody
• Performed the same as the first wash
• Notes about washes
• these can be highly variable - need to optimize
• Tip longer wash times or a greater number of
  washes can help reduce background

Example high background
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Signal Detection

• Depends on type of secondary conjugate used
• ECL/HRP add reagents and expose to film
• Enzymes add substrate
• Dyes (fluorescent/infrared) expose to
  fluorescent/infrared imager

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Stripping and reprobing

• Purpose of Stripping Blot
• To remove blocker, primary, and secondary
  antibodies that bound during previous probe
• Purpose of Reprobing Blot
• Detect same/different protein of interest on same
  blot using a different antibody
• Example use to quantitate phosphorylation
  differences
• Tip must test stripping efficiency
• Stripping will result in some loss of protein
  from your blot (10-30)
• Tip cannot strip some secondary antibodies
  (such as Alk. Phos. conjugated)

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Controls for Western Blots

• Loading controls
• Positive controls (ex purified protein of
  interest)
• Negative controls (ex sample known to lack
  protein of interest)
• Detection Controls
• Preincubate primary antibody with saturating
  amount of antigen
• Controls for quantitation
• standard curve of purified protein
• TIP essential to ensure that signal
• is in dynamic range of detection

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Antibody Screening Prep Gels

• Advantages
• Same protein loading on all strips
• Vertical strips, screen
• different antibodies
• Find optimal antibody concentration

3mm wide strips
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Other Resources

• Current Protocols Online
• excellent resource for general techniques
• Current Protocols in Molecular Biology
• Chapter 10, Analysis of Proteins
• Section II Electrophoretic Separation of
  Proteins
• Section III Detection of Proteins
• Detailed protocols buffers / reagents
• Chapter 11, Immunology
• Unit 11.2 ELISA
• Website
• http//www.mrw.interscience.wiley.com/cp/cpmb/cpmb
  _contents_fs.html

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• All following slides are for reference only, and
  will not be discussed in class

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Tank Transfer Basic protocol

• 1. Cut membrane to size of gel, mark one side
  with pencil for orientation
• 2. Wet nitrocellulose in transfer buffer
  
• (PVDF first wet thoroughly in 100 methanol,
  then wet in transfer buffer)
• 3. Assemble transfer apparatus
• 4. Run at constant voltage 30-35V overnight
  80-100V 1-4 hrs generally transfer performed in
  4C cold room

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Tank Transfer Basic Protocol

• Uses 1-5 liters of buffer (Tris/Glycine buffer or
  CAPS buffer)
• Can transfer multiple gels at a time with equal
  efficiency
• Can reuse the buffer in rig multiple times
• Better for difficult-to-transfer proteins (gt100
  kDa, hydrophobic)
• More time intensive

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Semidry Transfer Basic Protocol

• Cut membrane to size of gel, mark one side with
  pencil for orientation
• Wet nitrocellulose in transfer buffer (PVDF,
  first rinse in 100 methanol, then wet in
  transfer buffer)
• Assemble transfer apparatus
• Run at constant voltage 10-15V,10-30 min.
  (maximum 1hr), room temp.

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Blocking Basic protocol

• Dissolve nonfat dry milk or BSA in TTBS (Tris
  buffered saline with Tween-20) to give 2-10
  solution
• Immerse blot, put on rocker/shaker to provide
  constant agitation for at least 1 hour at room
  temperature
• Can incubate overnight _at_ 4C if necessary

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1 Antibody Basic protocol

• Blot can be cut into strips to probe with
  multiple antibodies simultaneously
• Ideal for detection of multiple proteins of
  different molecular weights in same blot
• Dilute antibody in TTBS or blocking buffer
• use appropriate dilution for each antibody (ex.
  1500, 11000)
• adding 1-5 milk may reduce background
• Primary antibodies usually expensive so to reduce
  volume, put blots in heat-sealable bags incubate
  room temp. 1-2hrs
• Can incubate overnight _at_ 4C if necessary (best
  for weak antibodies)

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2 Antibody Basic protocol

• Dilute antibody in TTBS
• use appropriate dilution for each antibody (ex.
  15,000 to 150,000)
• can add 1 milk to reduce background
• Generally less expensive than primary antibody,
  so use larger volumes incubate room temp. 45min.
  - 1hr.
• Tip Do not let this step go over 1hour
  (will increase background)

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Detection
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Detection Enhanced chemiluminescence (ECL)
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Detection ECL - Basic protocol

• After final wash, pour out TTBS, put blot
  protein-side-up on Saran Wrap on flat surface
• Generally you will have 2 solutions
• 1. luminol (the substrate) and an enhancer (ex.
  p-coumaric acid, p-iodophenol
• 2. hydrogen peroxide -- mix these well, apply to
  blot, incubate 1-5 minutes
• Remove excess solution (dab blot on Whatman
  paper)
• Place protein-side-down on clean piece of Saran
  Wrap, fold up, tape protein-side-up into gel
  cassette, expose to film

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Detection ECL

• Tips
• When using a commerically available ECL kit, the
  signal usually lasts longer than a homemade
  recipe
• Can also quantify signal from protein bands using
  a Fluor-S-Max machine
• Be sure to trace molecular weight standards on
  film before removing blot from cassette

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Stripping and reprobing Basic protocol

• Stripping Buffer
• 100mM b-mercaptoethanol, 2 SDS, 62.5mM Tris-HCl
  pH 6.8
• 1. Place membrane and stripping solution in
  heat-sealable bag
• 2. incubate at 50-70C for 30 minutes
• replace stripping buffer and incubate an
  additional 30 minutes
• rinse in TTBS