Blotting and Blotting and immunodetectionimmunodetection

MBV4020MBV4020Dept. of Molecular BiosciencesDept. of Molecular Biosciences

UiOUiOAutumn 2005Autumn 2005Winnie 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 the Here SDS is removed and methanol is introduced into the gelgel

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 0.5 For wet blotting membranes should be 0.5 cm longer and 0.5

cm wider than the gel.cm wider than the gel. For semi dry blotting the membrane must be same size as gel For semi dry blotting the membrane must be same size as gel

or smaller.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 larger For semi dry blotting the filter paper should be slightly larger

than the opening of the plastic shielding without exceeding than the opening of the plastic shielding without exceeding the size of the gel.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 membraneNitrocelluloseNitrocellulose PVDFPVDF

Polyvinylidene 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 adjustedThis buffer has a pH of approx. 8,3 and must not be adjusted 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 air in transfer buffer. Eliminate air bubbles.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 already blotting chamber which already contains transfer buffercontains transfer buffer

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 Large gel (15X21 cm): 1.0 A, approx. 100 V for 1-3 hrs at 15-2015-20oo 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 Place the blotting unit on a magnetic stirrer. This will ensure even temperatures and effective dissipation of heat. ensure 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 permanentpermanentlow low backgroundbackground

CoomassieCoomassieBlueBlue

1.5 ug1.5 ug NitrocellulosNitrocellulosee

PVDFPVDF permanentpermanenthigh high backgroundbackground

India InkIndia Ink 100 100 ngng

NitrocellulosNitrocellulosee

PVDFPVDF permanentpermanent

Biotin-AvidinBiotin-Avidin 30 ng30 ng NitrocellulosNitrocellulosee

PVDFPVDF permanentpermanentFades with Fades with timetime

ColloidalColloidalgoldgold

3 ng3 ng NitrocellulosNitrocellulosee

PVDFPVDF 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 saline, PBS, phosphate buffered saline,

pH 7.5-8.0pH 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

HorseradisHorseradishhperoxidaseperoxidase

200-500 pg200-500 pg DAB/NiClDAB/NiCl22 GoodGood NaNNaN33 inhibits inhibits HRP, HRP, endogenousendogenousPeroxidase Peroxidase act.act.

Alkaline Alkaline phosphatasphosphatasee

100 pg100 pg5 pg with5 pg withamplificatioamplificationn

BCIP/NBTBCIP/NBT GoodGood Endogenous Endogenous phosphatasephosphataseactivityactivity

ColloidalColloidalgoldgold

100 pg100 pg5 pg with5 pg withamplificatioamplificationn

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