Blotting and immunodetection MBV4020 Dept. of Molecular Biosciences UiO Autumn 2005 Winnie Eskild

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

Choices to be made Blotting Blotting Gel thicknes Gel thicknes Wet/semi dry Wet/semi dry Membrane type Membrane type Transfer conditions Transfer conditions Buffer Buffer Immunodetection Blocking Buffer Incubation time Antibody Washing Detection method

Blotting - wet or semi dry depends on protein type and size Proteins are transferred from the gel to a membrane by an electric field. Proteins are transferred from the gel to a membrane by an electric field. Proteins usually migrate towards the positive electrode Proteins usually migrate towards the positive electrode Protein type determines choice of method: Protein type determines choice of method: Hydrophobic or large proteins (>100 kDa) - wet blotting Hydrophobic or large proteins (>100 kDa) - wet blotting Transfer time up to 16 hours Transfer time up to 16 hours Hydrophilic or small proteins (<100 kDa) - semi dry blotting Hydrophilic or small proteins (<100 kDa) - semi dry blotting Transfer time up to 2 hours Transfer time up to 2 hours

Prepare gel for blotting Remove stacking gel Remove stacking gel Cut off a corner at the top of lane 1 Cut off a corner at the top of lane 1 Soak the gel in transfer buffer Soak the gel in transfer buffer Buffer contains methanol which makes the gel swell a little Buffer contains methanol which makes the gel swell a little Increases elution of SDS from the gel Increases elution of SDS from the gel Increases binding of proteins to the membrane 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 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 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 SDS helps protein migration out of the gel, but inhibits binding to the membrane

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

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.

Choice of membrane NitrocellulosePVDF Polyvinylidene difluoride Binding to membrane Hydrophobic interaction BackgroundLowHigh Special qualities Brittle after baking Works with SDS Physical characteristics Breaks easily Strong

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

Wet blotting Equilibrate gel in transfer buffer in separate tray. Equilibrate gel in transfer buffer in separate tray. Equilibrate filters and sponges in transfer buffer. Eliminate air bubbles. Equilibrate filters and sponges in transfer buffer. Eliminate air bubbles. PVDF membranes must be soaked in methanol, before equilibration in transfer buffer. 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

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

Semi dry blotting Equilibrate gel in transfer buffer in separate tray Equilibrate gel in transfer buffer in separate tray Six filters pr gel are soaked in transfer buffer Six filters pr gel are soaked in transfer buffer PVDF membranes must first be soaked in methanol, before equilibration 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

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

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

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.

Control of transfer Membrane staining: Several methods which vary with regard to sensitivity and reversibility Ponceau S 1-2 ug NitrocellulosePVDFreversible Amido Black 1.5 ug NitrocellulosePVDFpermanent low background CoomassieBlue 1.5 ug NitrocellulosePVDFpermanent high background India Ink 100 ng NitrocellulosePVDFpermanent Biotin-Avidin 30 ng NitrocellulosePVDFpermanent Fades with time Colloidalgold 3 ng NitrocellulosePVDFpermanent

Blocking Blocking reduces nonspesific binding of antibody (primary or secondary) to protein or membrane Blocking reduces nonspesific binding of antibody (primary or secondary) to protein or membrane Too little => high background Too little => high background Too much reduces the signal Too much reduces the signal Incubation time: Incubation time: 1-2 hrs at RT with shaking 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 TBS, TRIS-buffered saline, pH 7.5

Incubation with primary antibody Polyclonal (serum, IgG, affinity purified 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 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 Incubation time must be determined in each case Varies from 5 min at RT to ON at 4 o C Varies from 5 min at RT to ON at 4 o C Dilution must be determined individually in each case Dilution must be determined individually in each case Depends on titer and system sensitivity Depends on titer and system sensitivity Normal dilution for polyclonal: 1:1, :50,000 Normal dilution for polyclonal: 1:1, :50,000 Amplification of signal 2-10 times with biotin-streptavidin Amplification of signal 2-10 times with biotin-streptavidin

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

Incubation with secondary antibody Secondary antibody specificly recognizes IgG from the species where primary antibody was produced Secondary antibody specificly recognizes IgG from the species where primary antibody was produced Buffer: same as for primary antibody Buffer: same as for primary antibody Dilution must be determined in each case Dilution must be determined in each case Usually 1:1, :100,000 Usually 1:1, :100,000 Incubation time must be determined in each case Incubation time must be determined in each case Varies from 5 min to 2 hrs Varies from 5 min to 2 hrs enzym

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

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

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 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 Advantage: Very high sensitivity Disadvantage: More time consuming Disadvantage: More time consuming

Sensitivity MethodDetectionlimitSubstrate Stability of staining Limitations Horseradishperoxidase pg DAB/NiCl 2 Good NaN 3 inhibits HRP, endogenous Peroxidase act. Alkalinephosphatase 100 pg 5 pg with amplificationBCIP/NBTGood Endogenous phosphatase activity Colloidalgold 100 pg 5 pg with amplification0Good0 Background or loss of signal!!

Stripping and reprobing of Western filter Three methods 1. Incubate membrane for 30 min in 2% SDS, 100 mM TRIS pH 7.4, 100 mM  -mercapto ethanol at 70 o C. 2. Wash in H 2 O, incubate for 5 min in 0.2 M NaOH at RT. Wash in H 2 O and transfer to PBS. 3. Incubate for 10 min in 7 M guanidine-HCl at RT.Wash 3 times in TBS w/0.1% Tween 20