ABE Summer Workshop 2005 Southern & Western Blotting
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.
Southern Blot Process • Restriction digestion: breaks up DNA. • Gel run: separates DNA into bands. • Blot: transfer DNA from gel to nylon membrane. • Add probe: DNA complimentary to desired sequence labeled with DIG. • Add anti-DIG + AP, then substrate for chemiluminescence. • Expose to X-ray film, develop & print.
Our Initial Gel* * Before dropping.
Dig 2a-1 Und 2a-2 Empty Dig WT Plasmid Und 2a-1 Und 2b-2 Ladder Und WT Dig 2b-2 Dig 2a-2 Our Southern Blot Result
Und 2a-2 Dig 2a-1 Und 2a-2 Dig 2a-1 Empty Dig WT Dig 2b-2 Und 2b-2 Empty Ladder Und 2a-1 Plasmid Dig WT Und WT Dig 2b-2 Und 2a-1 Ladder Plasmid Dig 2a-2 Und 2b-2 Und WT Dig 2a-2 Gel & Blot Comparisons
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.
Protein Separation • Protein extraction: liquid N, grinding, buffer. • Spectrophotometer protein concentration assay for standardization of well loading. • Protein separation with 2 SDS-PAGE gels. • Visualization of gel results:a) Coomassie stain of all proteins.b) Western blot to identify specific PDI proteins.
Western Blot Process • Transfer proteins from PAGE to NC membrane. • Block with TBS and 5% nonfat milk. • React membrane with primary antibody to PDI-2 peptide (antibody made in rabbit). • Wash and react with secondary (donkey anti-rabbit) antibody conjugated to HRP. • Wash and react with substrate (luminol + enhancer.) Oxidized product results in light. • Light is detected with X-ray film. (Longer exposures appeared more effective.)
Western Blot Nitrocellulose membrane Polyacrylamide Gel
Protein Gel Comparisons 2B 2A WT
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.