Immunoblotting


Immunoblotting (PDF)


Immunoblotting is typically used to determine the amount (dot blot) and molecular weight (western blot) of an antigen present in a complex mixture. Assay sensitivity can be modulated by using different enzyme substrates and/or by using biotin-(strept)avidin conjugates in place of an enzyme conjugated secondary antibody. The highly sensitive procedure below is suggested. If decreased sensitivity is desired, replace the biotin-streptavidin conjugate with an antibody-peroxidase conjugate. All incubations are at room temperature.


This procedure is for nitrocellulose or PVDF membranes. Do not to touch the membrane! Wear gloves. Nylon membranes may be used, however, they are more difficult to block. To block nylon membranes use buffer without Tween-20, replace BSA with 10% nonfat dry milk and block for several hours to overnight at 37°C.


Reagents Required

  • Tris Buffered Saline with Tween-20. Use 10X TTBS, pH 7.5 (1.0 M Tris HCl, 1.5 M NaCl, 0.1% Tween-20) Code # MB-013. Dilute appropriate volume to 1X with deionized water. Store at room temperature up to one month.

  • TTBS with 1% BSA. To 100 ml of 1X TTBS add 1.0 g of Bovine Serum Albumin (BSA) Code # BSA-10. Dissolve and use immediately.

Procedure

  1. Transfer and immobilize antigen on nitrocellulose or PVDF membrane.

  2. Block by immersing the membrane in TTBS. Use just enough solution to cover the membrane. Never let the membrane become dry during the procedure. Incubate for 30 minutes with gentle agitation. The addition of 1.0% BSA to blocking solution increases signal-to-noise ratio over the use of TTBS alone. Some antigens and antibodies may be eluted in the presence of Tween-20. If this occurs, replace the Tween-20 with 1.0% BSA in all TTBS solutions and repeat the experiment.

  3. Transfer the membrane to diluted solution of primary antibody in TTBS. The appropriate dilution should be determined by trial and error. Serial ten fold dilutions starting at 1:10 are suggested. Incubate for 30 minutes with gentle agitation.

  4. Wash with 3 changes of TTBS for 5 minutes each with gentle agitation.

  5. Transfer the membrane to a dilute solution of biotinylated secondary antibody in TTBS. Confirm reagent specificity for primary antibody. Incubate for 30 minutes with gentle agitation.

  6. Repeat step 4.

  7. Transfer the membrane to a solution of peroxidase conjugated streptavidin (code# S000-03) appropriately diluted in TTBS. Incubate for 30 minutes with gentle agitation.

  8. During the above incubation, prepare the enzyme substrate. Use DAB (code DAB-10) for a dark brown color or TMB (code TMBM-100) for a bright blue color.

  9. Wash as in step 3.

  10. Transfer the membrane to the substrate solution. Incubate until color develops sufficiently (usually 2 to 20 minutes).

  11. Wash with 2 changes of water for 5 minutes each with gentle agitation. Allow membrane to dry and store in the dark.

References

  1. Antibodies, A Laboratory Manual. Ed Harlow and David Lane, eds. Cold Spring Harbor Press, 1988. Chapter 12 gives an excellent overview of Western blotting techniques, including India Ink staining.

  2. Molecular Cloning: A Laboratory Manual. 2nd Edition. J. Sambrook, E.F. Fritsch and T. Maniatis, eds. Cold Spring Harbor Press, 1989. Chapter 18 gives detailed protocols for the production of cell lysates, electrophoresis and blotting of proteins.

  3. Antibodies, A Practical Approach. 2nd Edition. Catty, D., ed. IRL Press, Oxford, England, 1990. Volumes I and II represent a detailed and complete reference for most current antibody techniques.