Blotting and immunodetection

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Blotting and immunodetection

• MBV4020
• Dept. of Molecular Biosciences
• UiO
• Autumn 2005
• Winnie Eskild

2

• Western technique analyses proteins after gel
  fractionation and transfer to a membrane.
• Advantage the proteins are fixed and accessible
  for analysis, in this case using an antibody.

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Choices to be made

• Blotting
• Gel thicknes
• Wet/semi dry
• Membrane type
• Transfer conditions
• Buffer

• Immunodetection
• Blocking
• Buffer
• Incubation time
• Antibody
• Washing
• Detection method

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Blotting - wet or semi drydepends on protein
type and size

• Proteins are transferred from the gel to a
  membrane by an electric field.
• Proteins usually migrate towards the positive
  electrode
• Protein type determines choice of method
• Hydrophobic or large proteins (gt100 kDa) - wet
  blotting
• Transfer time up to 16 hours
• Hydrophilic or small proteins (lt100 kDa) - semi
  dry blotting
• Transfer time up to 2 hours

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Prepare gel for blotting

• Remove stacking gel
• Cut off a corner at the top of lane 1
• Soak the gel in transfer buffer
• Buffer contains methanol which makes the gel
  swell a little
• Increases elution of SDS from the gel
• Increases binding of proteins to the membrane
• Soak for 15 min. Too little may lead to poor
  buffer equilibration Too much may lead to loss of
  proteins due to diffusion
• Here SDS is removed and methanol is introduced
  into the gel
• SDS helps protein migration out of the gel, but
  inhibits binding to the membrane

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Protein migration from gel to membrane

• SDS-denaturation leads to net negative charge
• SDS-denatured proteins migrate more easily out of
  the gel
• SDS left in the gel migrates to the membrane and
  binds to it gt competition with the protein
• Methanol facilitates eluation of SDS from gel and
  makes it swell a little
• Methanol detaches SDS from the protein gt
  increased binding of protein to the membrane
• Methanol reduces transfer efficiency due to
  renaturation of protein

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More preparation

• We need
• 1-2 membranes
• For wet blotting membranes should be 0.5 cm
  longer and 0.5 cm wider than the gel.
• For semi dry blotting the membrane must be same
  size as gel or smaller.
• 2-6 pcs filter paper (Whatman 3M)
• For wet blotting these should be slightly larger
  than the membrane but not exceed the size of
  blotting sponges.
• If sponges are worn thin use more filter paper.
• A too short distance to blotting sandwich will
  result in shadow pattern on the membrane.
• For semi dry blotting the filter paper should be
  slightly larger than the opening of the plastic
  shielding without exceeding the size of the gel.
• Blotting sponges/Scotch Brite
• Everything is soaked for min. 15 minutes.

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Choice of membrane
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Transferbuffer

• Standard buffer Towbin buffer
• 25 mM TRIS base
• 192 mM Glycine
• 0 - 0,2 SDS
• Increases transfer of proteins gt 60 kDa
• Reduces binding to membrane
• Cannot be used for nylon membranes
• 0 - 20 methanol
• Reduces transfer effectiveness
• Increases binding to membrane
• Note
• This buffer has a pH of approx. 8,3 and must not
  be adjusted
• pH adjustment introduces free ions which increase
  conductivity. Increased conductance ( mA) results
  in increased heat and thus risk of denaturation

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Wet blotting

• Equilibrate gel in transfer buffer in separate
  tray.
• Equilibrate filters and sponges in transfer
  buffer. Eliminate air bubbles.
• PVDF membranes must be soaked in methanol, before
  equilibration in transfer buffer.
• Nitrocellulose membranes are soaked directly in
  transfer buffer
• The transfer sandwich is packed under buffer as
  shown in the figure.
• Roll a glass rod over each layer to remove air
  bubbles (inhibit transfer of proteins)
• Mount transfer sandwich in blotting chamber which
  already contains transfer buffer

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Blotting conditions - wet transfer

• Here amperes and voltage are determined by gel
  size
• Mini gels (9X10 cm) 200 mA, approx. 50 V for 2
  hrs at 15-20o C
• or 400 mA, approx. 100 V for 1 hr at 15-20o C
• Large gel (15X21 cm) 1.0 A, approx. 100 V for
  1-3 hrs at 15-20o C
• Alternatively Overnight blotting at 15-25 V in
  cold room
• Place the blotting unit on a magnetic stirrer.
  This will ensure even temperatures and effective
  dissipation of heat.

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Semi dry blotting

• Equilibrate gel in transfer buffer in separate
  tray
• Six filters pr gel are soaked in transfer
  buffer
• PVDF membranes must first be soaked in methanol,
  before equilibration in transfer buffer.
• Nitrocellulose membranes may be soaked in
  transfer buffer directly
• Place plastic shielding on lower electrode ()
  with opening in the centre

• Pack sandwich as shown in the figure
• Roll a glass rod over each layer to remove
  air-bubbles
• The whole sandwich should be saturated with
  buffer
• Place upper electrode (-). Ensure good contact
  over the transfer area

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Blotting conditions - semidry blotting

• Never use more than 0.8 mA/cm2. Calculation of
  this is based on the opening area in the plastic
  shielding, which is slightly smaller than the
  gel.
• Blotting should not exceed 2 hrs. Heat production
  dries the filter.

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Control of transfer

• Gel stainingIncubate approx. 2 hrs in Coomassie
  Blue staining solution and destain for 1 hr
• Large proteins are difficult to transfer.
• Always some residues left in gel

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Detection

• Western technique analyses proteins after gel
  fractionation and transfer to a membrane.
• Advantage the proteins are fixed and accessible
  for analysis, in this case using an antibody.

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Control of transferMembrane staining Several
methods which vary with regard to sensitivity and
reversibility
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Blocking

• Blocking reduces nonspesific binding of antibody
  (primary or secondary) to protein or membrane
• Too little gt high background
• Too much reduces the signal
• Incubation time
• 1-2 hrs at RT with shaking

• Many different blocking agents
• Fat free dry milk
• Tween 20
• Bovin serum albumin
• Casein
• Gelatin
• Hemoglobin
• Ovalbumin
• Buffer
• PBS, phosphate buffered saline, pH 7.5-8.0
• TBS, TRIS-buffered saline, pH 7.5

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Incubation with primary antibody

• Polyclonal (serum, IgG, affinity purified
  antibody)
• or monoclonal (ascites, cell supernatant,
  affinity purified antibody) may be used.
• Buffer is often the same as for blocking or even
  just PBS w/Tween 20 or TBS w/Tween 20
• Incubation time must be determined in each case
• Varies from 5 min at RT to ON at 4oC
• Dilution must be determined individually in each
  case
• Depends on titer and system sensitivity
• Normal dilution for polyclonal 11,000 -
  150,000
• Amplification of signal 2-10 times with
  biotin-streptavidin

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Washing

• Buffer PBS w/Tween 20 or TBS w/Tween 20
• TW20 concentration must be determined for each
  antibody and antigen
• Usually 0.01-0.2
• Time Number of washes and duration of each wash
  must be determined in each case
• Usually 3X5 min 3X15 min
• Use large buffer volume 50-100 ml for 8X10 cm
  membrane
• Incubation with vigorous shaking

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Incubation with secondary antibody

• Secondary antibody specificly recognizes IgG from
  the species where primary antibody was produced
• Buffer same as for primary antibody
• Dilution must be determined in each case
• Usually 11,000 - 1100,000
• Incubation time must be determined in each case
• Varies from 5 min to 2 hrs

enzym
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Detection Direct, indirect or with
biotin-streptavidin amplification

• Direct detection
• Enzyme alkaline phosphatase, horse radish
  peroxidase
• Gray-black precipitate or chemiluminiscence
• Radioactive ligand 125I
• is coupled to the primary antibody.
• Almost direct detection
• Biotin binds streptavidin coupled to enzyme (AP,
  HRP)
• is coupled to the primary antibody.
• Advantages Quick, low background
• Disadvantages Lower sensitivity, more work to
  purify and label antibody

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Detection Indirect

• Indirect detection Here we use a secondary
  antibody directed against IgG from the species
  where the primary antibody was made
• Secondary antibody carries a label radioactive
  ligand, biotin or enzyme (AP, HRP)
• Advantages same secondary antibody for all
  primary antibodies from one species, increased
  sensitivity
• Disadvantages Somewhat higher background, takes
  a little longer

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Detection With biotin-streptavidin amplification

• Biotin-streptavidin amplification Here a biotin
  molecule is coupled to the secondary antibody. A
  complex of streptavidin and enzyme is added. This
  method results in many more enzyme molecules pr
  secondary antibody molecule
• Advantage Very high sensitivity
• Disadvantage More time consuming

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Sensitivity
Background or loss of signal!!
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Stripping and reprobing of Western filterThree
methods

• Incubate membrane for 30 min in 2 SDS, 100 mM
  TRIS pH 7.4, 100 mM ?-mercapto ethanol at 70oC.
• Wash in H2O, incubate for 5 min in 0.2 M NaOH at
  RT. Wash in H2O and transfer to PBS.
• Incubate for 10 min in 7 M guanidine-HCl at
  RT.Wash 3 times in TBS w/0.1 Tween 20