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Blotting and immunodetection

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. Advantage: the proteins are fixed and accessible for analysis, in this case using an antibody.

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Blotting and immunodetection

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  1. 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.

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

  4. Blotting - wet or semi drydepends onprotein 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 (>100 kDa) - wet blotting • Transfer time up to 16 hours • Hydrophilic or small proteins (<100 kDa) - semi dry blotting • Transfer time up to 2 hours

  5. 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

  6. 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 => competition with the protein • 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 reduces transfer efficiency due to renaturation of protein

  7. 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.

  8. Choice of membrane

  9. Transferbuffer • Standard buffer: Towbin buffer: • 25 mM TRIS base • 192 mM Glycine • 0 - 0,2% SDS • Increases transfer of proteins > 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

  10. 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

  11. 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 15-20oC • 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.

  12. 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 Semi dry blotting

  13. 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.

  14. Control of transfer • Gel staining:Incubate 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

  15. 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.

  16. Control of transferMembrane staining: Several methods which vary with regard to sensitivity and reversibility

  17. Blocking reduces nonspesific binding of antibody (primary or secondary) to protein or membrane Too little => 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 Blocking

  18. 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: 1:1,000 - 1:50,000 • Amplification of signal 2-10 times with biotin-streptavidin

  19. 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

  20. 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 1:1,000 - 1:100,000 • Incubation time must be determined in each case • Varies from 5 min to 2 hrs enzym

  21. 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

  22. 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

  23. 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

  24. Sensitivity Background or loss of signal!!

  25. 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

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