Blotting and immunodetection MBV4020 Dept. of Molecular Biosciences UiO Autumn 2005 Winnie Eskild.
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Transcript of Blotting and immunodetection MBV4020 Dept. of Molecular Biosciences UiO Autumn 2005 Winnie Eskild.
Blotting and Blotting and immunodetectionimmunodetection
MBV4020MBV4020
Dept. of Molecular BiosciencesDept. of Molecular Biosciences
UiOUiO
Autumn 2005Autumn 2005
Winnie EskildWinnie Eskild
Western technique analyses proteins Western technique analyses proteins after gel fractionation and transfer after gel fractionation and transfer to a membrane.to a membrane.
Advantage: the proteins are fixed Advantage: the proteins are fixed and accessible for analysis, in this and accessible for analysis, in this case using an antibody.case using an antibody.
Choices to be madeChoices to be made
BlottingBlotting Gel thicknesGel thicknes Wet/semi dryWet/semi dry Membrane typeMembrane type Transfer conditionsTransfer conditions BufferBuffer
ImmunodetectionImmunodetection BlockingBlocking BufferBuffer Incubation timeIncubation time AntibodyAntibody WashingWashing Detection methodDetection method
Blotting - wet or semi dryBlotting - wet or semi drydepends ondepends on protein type and sizeprotein type and size
Proteins are transferred Proteins are transferred from the gel to a membrane from the gel to a membrane by an electric field.by an electric field.
Proteins usually migrate Proteins usually migrate towards the positive towards the positive electrodeelectrode
Protein type determines Protein type determines choice of method:choice of method: Hydrophobic or large proteins Hydrophobic or large proteins
(>100 kDa) - wet blotting(>100 kDa) - wet blotting Transfer time up to 16 hoursTransfer time up to 16 hours
Hydrophilic or small proteins Hydrophilic or small proteins (<100 kDa) - semi dry blotting(<100 kDa) - semi dry blotting
Transfer time up to 2 hoursTransfer time up to 2 hours
Prepare gel for blottingPrepare gel for blotting
Remove stacking gelRemove stacking gel Cut off a corner at the top of lane 1Cut off a corner at the top of lane 1 Soak the gel in transfer bufferSoak the gel in transfer buffer
Buffer contains methanol which makes the gel swell a littleBuffer contains methanol which makes the gel swell a little Increases elution of SDS from the gelIncreases elution of SDS from the gel Increases binding of proteins to the membraneIncreases binding of proteins to the membrane
Soak for 15 min. Too little may lead to poor buffer Soak for 15 min. Too little may lead to poor buffer equilibration Too much may lead to loss of proteins due to equilibration Too much may lead to loss of proteins due to diffusiondiffusion
Here SDS is removed and methanol is introduced into Here SDS is removed and methanol is introduced into the gelthe gel
SDS helps protein migration out of the gel, but inhibits SDS helps protein migration out of the gel, but inhibits binding to the membranebinding to the membrane
Protein migration from gel to Protein migration from gel to membranemembrane
SDS-denaturation leads to net negative chargeSDS-denaturation leads to net negative charge SDS-denatured proteins migrate more easily out of SDS-denatured proteins migrate more easily out of
the gelthe gel SDS left in the gel migrates to the membrane and SDS left in the gel migrates to the membrane and
binds to it => competition with the proteinbinds to it => competition with the protein Methanol facilitates eluation of SDS from gel and Methanol facilitates eluation of SDS from gel and
makes it swell a littlemakes it swell a little Methanol detaches SDS from the protein => Methanol detaches SDS from the protein =>
increased binding of protein to the membraneincreased binding of protein to the membrane Methanol reduces transfer efficiency due to Methanol reduces transfer efficiency due to
renaturation of proteinrenaturation of protein
More preparationMore preparation We need: We need:
1-2 membranes1-2 membranes For wet blotting membranes should be 0.5 cm longer and For wet blotting membranes should be 0.5 cm longer and
0.5 cm wider than the gel.0.5 cm wider than the gel. For semi dry blotting the membrane must be same size as For semi dry blotting the membrane must be same size as
gel or smaller.gel or smaller. 2-6 pcs filter paper (Whatman 3M)2-6 pcs filter paper (Whatman 3M)
For wet blotting these should be slightly larger than the For wet blotting these should be slightly larger than the membrane but not exceed the size of blotting sponges.membrane but not exceed the size of blotting sponges.
If sponges are worn thin use more filter paper.If sponges are worn thin use more filter paper. A too short distance to blotting sandwich will result in A too short distance to blotting sandwich will result in
”shadow pattern” on the membrane.”shadow pattern” on the membrane. For semi dry blotting the filter paper should be slightly For semi dry blotting the filter paper should be slightly
larger than the opening of the plastic shielding without larger than the opening of the plastic shielding without exceeding the size of the gel.exceeding the size of the gel.
Blotting sponges/Scotch BriteBlotting sponges/Scotch Brite Everything is soaked for min. 15 minutes.Everything is soaked for min. 15 minutes.
Choice of membraneChoice of membrane
NitrocelluloseNitrocellulose PVDFPVDFPolyvinylidene difluoridePolyvinylidene difluoride
Binding to Binding to membranemembrane
Hydrophobic Hydrophobic interactioninteraction
Hydrophobic Hydrophobic interactioninteraction
BackgroundBackground LowLow HighHigh
Special qualitiesSpecial qualities Brittle after bakingBrittle after baking Works with SDSWorks with SDS
Physical Physical characteristicscharacteristics
Breaks easilyBreaks easily StrongStrong
TransferbufferTransferbuffer Standard buffer: Towbin buffer:Standard buffer: Towbin buffer:
25 mM TRIS base25 mM TRIS base 192 mM Glycine192 mM Glycine 0 - 0,2% SDS0 - 0,2% SDS
Increases transfer of proteins > 60 kDaIncreases transfer of proteins > 60 kDa Reduces binding to membraneReduces binding to membrane Cannot be used for nylon membranesCannot be used for nylon membranes
0 - 20% methanol0 - 20% methanol Reduces transfer effectivenessReduces transfer effectiveness Increases binding to membraneIncreases binding to membrane
Note:Note: This buffer has a pH of approx. 8,3 and must not be This buffer has a pH of approx. 8,3 and must not be
adjustedadjusted pH adjustment introduces free ions which increase pH adjustment introduces free ions which increase
conductivity. Increased conductance ( mA) results in conductivity. Increased conductance ( mA) results in increased heat and thus risk of denaturationincreased heat and thus risk of denaturation
Wet blottingWet blotting Equilibrate gel in transfer Equilibrate gel in transfer
buffer in separate tray.buffer in separate tray. Equilibrate filters and sponges Equilibrate filters and sponges
in transfer buffer. Eliminate in transfer buffer. Eliminate air bubbles.air bubbles.
PVDF membranes must be PVDF membranes must be soaked in methanol, before soaked in methanol, before equilibration in transfer buffer. equilibration in transfer buffer.
Nitrocellulose membranes are Nitrocellulose membranes are soaked directly in transfer soaked directly in transfer bufferbuffer
The transfer sandwich is The transfer sandwich is packed under buffer as shown packed under buffer as shown in the figure.in the figure.
Roll a glass rod over each Roll a glass rod over each layer to remove air bubbles layer to remove air bubbles (inhibit transfer of proteins)(inhibit transfer of proteins)
Mount transfer sandwich in Mount transfer sandwich in blotting chamber which blotting chamber which already contains transfer already contains transfer bufferbuffer
Blotting conditions - wet transferBlotting conditions - wet transfer
Here amperes and voltage are determined by gel sizeHere amperes and voltage are determined by gel size Mini gels (9X10 cm): 200 mA, approx. 50 V for 2 hrs at 15-Mini gels (9X10 cm): 200 mA, approx. 50 V for 2 hrs at 15-
2020oo C C oror 400 mA, approx. 100 V for 1 hr at 15-20400 mA, approx. 100 V for 1 hr at 15-20oo CC
Large gel (15X21 cm): 1.0 A, approx. 100 V for 1-3 hrs at 15-Large gel (15X21 cm): 1.0 A, approx. 100 V for 1-3 hrs at 15-
2020oo C C
Alternatively: Overnight blotting at 15-25 V in cold roomAlternatively: Overnight blotting at 15-25 V in cold room
Place the blotting unit on a magnetic stirrer. This will ensure Place the blotting unit on a magnetic stirrer. This will ensure even temperatures and effective dissipation of heat. even temperatures and effective dissipation of heat.
Semi dry blottingSemi dry blotting Equilibrate gel in transfer buffer in Equilibrate gel in transfer buffer in
separate trayseparate tray Six filters pr gel are soaked in Six filters pr gel are soaked in
transfer buffertransfer buffer PVDF membranes must first be PVDF membranes must first be
soaked in methanol, before soaked in methanol, before equilibration in transfer buffer. equilibration in transfer buffer. Nitrocellulose membranes may be Nitrocellulose membranes may be soaked in transfer buffer directlysoaked in transfer buffer directly
Place plastic shielding on lower Place plastic shielding on lower electrode (+) with opening in the electrode (+) with opening in the centrecentre
Pack sandwich as shown in the figurePack sandwich as shown in the figure Roll a glass rod over each layer to remove air-bubblesRoll a glass rod over each layer to remove air-bubbles The whole sandwich should be saturated with bufferThe whole sandwich should be saturated with buffer Place upper electrode (-). Ensure good contact over the transfer areaPlace upper electrode (-). Ensure good contact over the transfer area
Blotting conditions - semidry Blotting conditions - semidry blottingblotting
Never use more than 0.8 mA/cmNever use more than 0.8 mA/cm22. Calculation of this . Calculation of this is based on the opening area in the plastic shielding, is based on the opening area in the plastic shielding, which is slightly smaller than the gel.which is slightly smaller than the gel.
Blotting should not exceed 2 hrs. Heat production Blotting should not exceed 2 hrs. Heat production dries the filter.dries the filter.
Control of transferControl of transfer
Gel staining:Gel staining:Incubate approx. 2 hrs in Coomassie Blue staining Incubate approx. 2 hrs in Coomassie Blue staining solution and destain for 1 hrsolution and destain for 1 hr
Large proteins are difficult to transfer.Large proteins are difficult to transfer. Always some residues left in gelAlways some residues left in gel
DetectionDetection
Western technique Western technique analyses proteins analyses proteins after gel after gel fractionation and fractionation and transfer to a transfer to a membrane.membrane.
Advantage: the Advantage: the proteins are fixed proteins are fixed and accessible for and accessible for analysis, in this analysis, in this case using an case using an antibody.antibody.
Control of transferControl of transferMembrane staining: Several methods which Membrane staining: Several methods which vary with regard to sensitivity and reversibilityvary with regard to sensitivity and reversibility
Ponceau SPonceau S 1-2 ug1-2 ug NitrocellulosNitrocellulosee
PVDFPVDF reversiblereversible
Amido BlackAmido Black 1.5 ug1.5 ug NitrocellulosNitrocellulosee
PVDFPVDF permanentpermanent
low low backgroundbackground
CoomassieCoomassie
BlueBlue1.5 ug1.5 ug NitrocellulosNitrocellulos
eePVDFPVDF permanentpermanent
high high backgroundbackground
India InkIndia Ink 100 100 ngng
NitrocellulosNitrocellulosee
PVDFPVDF permanentpermanent
Biotin-AvidinBiotin-Avidin 30 ng30 ng NitrocellulosNitrocellulosee
PVDFPVDF permanentpermanent
Fades with Fades with timetime
ColloidalColloidal
goldgold3 ng3 ng NitrocellulosNitrocellulos
eePVDFPVDF permanentpermanent
BlockingBlocking
Blocking reduces Blocking reduces nonspesific binding of nonspesific binding of antibody (primary or antibody (primary or secondary) to protein or secondary) to protein or membranemembrane
Too little => high Too little => high backgroundbackground
Too much reduces the Too much reduces the signalsignal
Incubation time:Incubation time: 1-2 hrs at RT with shaking1-2 hrs at RT with shaking
Many different blocking Many different blocking agentsagents Fat free dry milkFat free dry milk Tween 20Tween 20 Bovin serum albuminBovin serum albumin CaseinCasein GelatinGelatin HemoglobinHemoglobin OvalbuminOvalbumin
Buffer:Buffer: PBS, phosphate buffered PBS, phosphate buffered
saline, pH 7.5-8.0saline, pH 7.5-8.0 TBS, TRIS-buffered saline, pH TBS, TRIS-buffered saline, pH
7.57.5
Incubation with primary antibody Incubation with primary antibody
Polyclonal (serum, IgG, affinity purified antibody) Polyclonal (serum, IgG, affinity purified antibody)
or monoclonal (ascites, cell supernatant, affinity or monoclonal (ascites, cell supernatant, affinity purified antibody) may be used.purified antibody) may be used.
Buffer is often the same as for blocking or even Buffer is often the same as for blocking or even just PBS w/Tween 20 or TBS w/Tween 20just PBS w/Tween 20 or TBS w/Tween 20
Incubation time must be determined in each case Incubation time must be determined in each case Varies from 5 min at RT to ON at 4Varies from 5 min at RT to ON at 4ooCC
Dilution must be determined individually in each Dilution must be determined individually in each casecase Depends on titer and system sensitivityDepends on titer and system sensitivity Normal dilution for polyclonal: 1:1,000 - 1:50,000Normal dilution for polyclonal: 1:1,000 - 1:50,000 Amplification of signal 2-10 times with biotin-Amplification of signal 2-10 times with biotin-
streptavidinstreptavidin
WashingWashing
Buffer: PBS w/Tween 20 or TBS w/Tween 20Buffer: PBS w/Tween 20 or TBS w/Tween 20 TW20 concentration must be determined for each TW20 concentration must be determined for each
antibody and antigenantibody and antigen Usually 0.01-0.2%Usually 0.01-0.2%
Time: Number of washes and duration of each Time: Number of washes and duration of each wash must be determined in each casewash must be determined in each case Usually 3X5 min + 3X15 minUsually 3X5 min + 3X15 min Use large buffer volume: 50-100 ml for 8X10 cm Use large buffer volume: 50-100 ml for 8X10 cm
membranemembrane Incubation with vigorous shakingIncubation with vigorous shaking
Incubation with secondary Incubation with secondary antibodyantibody
Secondary antibody specificly recognizes IgG Secondary antibody specificly recognizes IgG from the species where primary antibody was from the species where primary antibody was producedproduced
Buffer: same as for primary antibodyBuffer: same as for primary antibody Dilution must be determined in each caseDilution must be determined in each case
Usually 1:1,000 - 1:100,000Usually 1:1,000 - 1:100,000 Incubation time must be determined in each caseIncubation time must be determined in each case
Varies from 5 min to 2 hrsVaries from 5 min to 2 hrs
enzym
Detection Detection Direct, indirect or with biotin-streptavidin Direct, indirect or with biotin-streptavidin
amplificationamplification Direct detection: Direct detection:
Enzyme: alkaline phosphatase, horse radish peroxidase Enzyme: alkaline phosphatase, horse radish peroxidase Gray-black precipitate or chemiluminiscenceGray-black precipitate or chemiluminiscence
Radioactive ligand: Radioactive ligand: 125125I I
is coupled to the primary antibody.is coupled to the primary antibody. Almost direct detection: Almost direct detection:
Biotin: binds streptavidin coupled to enzyme (AP, HRP)Biotin: binds streptavidin coupled to enzyme (AP, HRP)
is coupled to the primary antibody.is coupled to the primary antibody.
Advantages: Quick, low backgroundAdvantages: Quick, low background Disadvantages: Lower sensitivity, more work to Disadvantages: Lower sensitivity, more work to
purify and label antibodypurify and label antibody
Detection Detection IndirectIndirect
Indirect detection: Here we use a secondary Indirect detection: Here we use a secondary antibody directed against IgG from the species antibody directed against IgG from the species where the primary antibody was madewhere the primary antibody was made
Secondary antibody carries a label: radioactive Secondary antibody carries a label: radioactive ligand, biotin or enzyme (AP, HRP)ligand, biotin or enzyme (AP, HRP)
Advantages: same secondary antibody for all Advantages: same secondary antibody for all primary antibodies from one species, increased primary antibodies from one species, increased sensitivitysensitivity
Disadvantages: Somewhat higher background, Disadvantages: Somewhat higher background, takes a little longertakes a little longer
Detection Detection With biotin-streptavidin amplificationWith biotin-streptavidin amplification
Biotin-streptavidin amplification: Here a biotin Biotin-streptavidin amplification: Here a biotin molecule is coupled to the secondary antibody. A molecule is coupled to the secondary antibody. A complex of streptavidin and enzyme is added. This complex of streptavidin and enzyme is added. This method results in many more enzyme molecules pr method results in many more enzyme molecules pr secondary antibody moleculesecondary antibody molecule
Advantage: Very high sensitivityAdvantage: Very high sensitivity Disadvantage: More time consumingDisadvantage: More time consuming
Sensitivity Sensitivity
MethodMethod DetectionDetection
limitlimitSubstrateSubstrate StabilitStabilit
y of y of staininstainingg
LimitationsLimitations
HorseradisHorseradishh
peroxidaseperoxidase
200-500 pg200-500 pg DAB/NiClDAB/NiCl22 GoodGood NaNNaN33 inhibits inhibits
HRP, HRP, endogenousendogenous
Peroxidase Peroxidase act.act.
Alkaline Alkaline
phosphatasphosphatasee
100 pg100 pg
5 pg with5 pg with
amplificatioamplificationn
BCIP/NBTBCIP/NBT GoodGood Endogenous Endogenous phosphatasephosphatase
activityactivity
ColloidalColloidal
goldgold100 pg100 pg
5 pg with5 pg with
amplificatioamplificationn
00 GoodGood 00Background or loss of signal!!
Stripping and reprobing of Stripping and reprobing of Western filterWestern filterThree methodsThree methods
1.1. Incubate membrane for 30 min in 2% SDS, 100 Incubate membrane for 30 min in 2% SDS, 100 mM TRIS pH 7.4, 100 mM mM TRIS pH 7.4, 100 mM -mercapto ethanol at -mercapto ethanol at 7070ooC.C.
2.2. Wash in HWash in H22O, incubate for 5 min in 0.2 M NaOH O, incubate for 5 min in 0.2 M NaOH
at RT. Wash in Hat RT. Wash in H22O and transfer to PBS.O and transfer to PBS.
3.3. Incubate for 10 min in 7 M guanidine-HCl at Incubate for 10 min in 7 M guanidine-HCl at RT.Wash 3 times in TBS w/0.1% Tween 20RT.Wash 3 times in TBS w/0.1% Tween 20