Transcript SouthernHybridization - University of Hawaii

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
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.
Restriction Digestion for
Southern Blot
• Wild Type (Genomic)
• PDI Plasmid PDI-2
• PDI Genomic Mutants:
1. 2A-1
1. 2A-1
1. 7A-1
2. 2A-2
2. 2A-2
2. 7B-1
3. 2B-2
3. 2B-2
• Restriction Enzymes:
3. 7B-2
EcoR1
HindIII
EcoR1
Our Initial Gel*
* Before dropping.
Our Southern Blot Result
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
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.
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.)
Western Blot
Nitrocellulose membrane
Polyacrylamide Gel
Our Coomassie stain result
Our Western blot result
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.