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 3. 7B-2

• Restriction Enzymes:

EcoR1 HindIII EcoR1

Our Initial Gel*

* Before dropping.

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

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

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

Polyacrylamide Gel

Nitrocellulose membrane

Our Coomassie stain result

Our Western blot result

Protein Gel ComparisonsWT2A2B

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.