abe summer workshop 2005 southern & western blotting
TRANSCRIPT
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ABE Summer Workshop 2005
Southern & Western Blotting
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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.
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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.
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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 3. 7B-2
• Restriction Enzymes:
EcoR1 HindIII EcoR1
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Our Initial Gel*
* Before dropping.
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Our Southern Blot Result
Em
p ty
Pla
smid
Dig
2b-
2
Dig
WT
Dig
2a-
1
Dig
2a-
2
Un d
2b -
2
Lad
der
Un d
WT
Un d
2a -
2
Un d
2a -
1
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Gel & Blot Comparisons
Em
p ty
Pla
smid
Dig
2b-
2
Dig
WT
Dig
2a-
1
Dig
2a-
2
Un d
2b -
2
Lad
der
Un d
WT
Un d
2a -
2
Un d
2a -
1
Em
p ty
Pla
smid
Dig
2b-
2
Dig
WT
Dig
2a-
1
Dig
2a-
2
Un d
2b -
2
Lad
der
Un d
WT
Un d
2a -
2
Un d
2a -
1
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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.
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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.
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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.)
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Western Blot
Polyacrylamide Gel
Nitrocellulose membrane
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Our Coomassie stain result
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Our Western blot result
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Protein Gel ComparisonsWT2A2B
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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.