1. ABE Summer Workshop 2005 Southern & Western Blotting

2. Goals with Southern Blot Using specific PDI gene probes: Identify PDI genes in wild type Arabidopsis plants. Determine the status of PDI genes in T-DNA Arabidopsis mutants.

3. Southern Blot Process 1.Restriction digestion: breaks up DNA. 2.Gel run: separates DNA into bands. 3.Blot: transfer DNA from gel to nylon membrane. 4.Add probe: DNA complimentary to desired sequence labeled with DIG. 5.Add anti-DIG + AP, then substrate for chemiluminescence. 6.Expose to X-ray film, develop & print.

4. Restriction Digestion for Southern Blot Wild Type (Genomic) PDI Plasmid PDI-2 PDI Genomic Mutants: 1. 2A-1 1. 7A-1 2. 2A-2 2. 7B-1 3. 2B-2 3. 7B-2 Restriction Enzymes: EcoR1 HindIIIEcoR1

5. Our Initial Gel* * Before dropping.

6. Our Southern Blot Result Empty Plasmid Dig 2b-2 Dig WT Dig 2a-1 Dig 2a-2 Und 2b-2 Ladder Und WT Und 2a-2 Und 2a-1

7. Gel & Blot Comparisons Empty Plasmid Dig 2b-2 Dig WT Dig 2a-1 Dig 2a-2 Und 2b-2 Ladder Und WT Und 2a-2 Und 2a-1 Empty Plasmid Dig 2b-2 Dig WT Dig 2a-1 Dig 2a-2 Und 2b-2 Ladder Und WT Und 2a-2 Und 2a-1

8. Goals with Western Blot Using antibodies specific to Arabidopsis PDI proteins: Detect PDI protein in wild type plants. In mutant plants, determine the effect of the T-DNA insert on the expression of the PDI gene through movement or deletion of PDI protein band.

9. Protein Separation 1.Protein extraction: liquid N, grinding, buffer. 2.Spectrophotometer protein concentration assay for standardization of well loading. 3.Protein separation with 2 SDS-PAGE gels. 4.Visualization of gel results: a) Coomassie stain of all proteins. b) Western blot to identify specific PDI proteins.

10. Western Blot Process 1.Transfer proteins from PAGE to NC membrane. 2.Block with TBS and 5% nonfat milk. 3.React membrane with primary antibody to PDI-2 peptide (antibody made in rabbit). 4.Wash and react with secondary (donkey anti-rabbit) antibody conjugated to HRP. 5.Wash and react with substrate (luminol + enhancer.) Oxidized product results in light. 6.Light is detected with X-ray film. (Longer exposures appeared more effective.)

11. Western Blot Polyacrylamide Gel Nitrocellulose membrane

12. Our Coomassie stain result

13. Our Western blot result

14. Protein Gel Comparisons WT2A2B

15. Western Blot Interpretation Bands displayed on our blot are ambiguous. We have 3 alternative explanations: a) All bands in all lanes are alternative forms of PDI-2. b) Anti-PDI peptide antibody from rabbit reacts with similar epitopes on unrelated proteins. c) 2° antibody from donkey reacts to similar epitopes on unrelated proteins.