1. Research Techniques Made Simple: Blotting M.W. Nicholas, MD, PhD 1 and Kelly Nelson, MD 2 1 University of North Carolina at Chapel Hill and 2 Duke University Departments of Dermatology

2. Blotting Techniques Allows detection of specific biomolecules in a sample (DNA or RNA sequences or protein) Southern blot (detection of DNA sequences) first developed by Dr. Southern in 1975 Subsequently adapted to detect other molecules – Northern blot: RNA sequences – Western blot: proteins

3. Blotting Techniques Three step process: 1.Gel electrophoresis separates molecules by size and charge 2.Transfer separated molecules to a membrane (maintaining previous separation) 3.Treat with labeled probe, which binds molecule of interest and allows for detection. When antibodies are used, an unlabeled antibody that recognizes the target is used first (primary), followed by a labeled antibody directed against the Fc portion of the probe antibody (secondary)

4. Step 1: Target molecules vertically separated by size/charge using gel electrophoresis Step 2: Target molecules horizontally transferred to membrane Step 3: Labeled probe confirms molecule of interest Ladder Primary antibody Labeled secondary antibody Blotting: A Three-Step Process

5. Other Applications of Blotting The western technique may also be used to identify targets of circulating autoantibodies in serum of patients Target proteins are blotted to the membrane, which is then treated with patient serum and an anti-human Fc labeled probe This method identified targets in paraneoplastic pemphigus and is used clinically to confirm presence of anti-collagen VII antibodies in patients with epidermolysis bullosa acquisita

6. Blotting Techniques Southern and northern blots have been replaced by real-time PCR techniques and fluorescent in situ hybridization (FISH) Western blots still used in some settings, but alternative techniques used more commonly: – Enzyme linked immunosorbent assay (ELISA) – Flow cytometry – Immunohistochemistry – Immunofluorescence

7. Western Blot Limitations Proteins must be denatured (unfolded) for western blotting This can disrupt the three dimensional (tertiary) structure recognized by the antibody probe (epitope)

8. Blotting Techniques NameTargetProbes Used Newer alternative techniques SouthernDNA Complementary (antisense) sequence of DNA or RNA PCR, real-time PCR, FISH NorthernRNA Complementary sequence of DNA or RNA RT-PCR, real-time RT-PCR WesternProteinMonoclonal antibodyELISA, immunohistochemistry, immunofluorescence, flow cytometry