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ABE Summer Workshop 2005

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.

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ABE Summer Workshop 2005

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  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 • 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.

  4. Restriction Digestion for Southern Blot

  5. Our Initial Gel* * Before dropping.

  6. 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

  7. 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

  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 • 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.

  10. 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.)

  11. Western Blot Nitrocellulose membrane Polyacrylamide Gel

  12. Our Coomassie stain result

  13. Our Western blot result

  14. Protein Gel Comparisons 2B 2A WT

  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.

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