Protocol S.3

Southwestern Blot

This protocol is adopted from the Lambda gt22a screening protocol (L.1) and the protein gels protocol P.1.

Solutions

 

10X Klenow Buffer

0.5 M Tris 7.5 500 ml 1M Tris 7.5

0.1 M MgCl2 100 ml 1M MgCl2

10 mM DTT 100 ml 0.1 M DTT

0.5 mg/ml BSA 50 ml 10 mg/ml BSA

250 ml Q

 

20X dNTP mix

10 mM dGTP 3 ml 100 mM dGTP

10 mM dTTP 3 ml 100 mM dTTP

24 ml Q

 

Calf Thymus DNA

Sonicate the entire bottle in 25 ml Q

Pass through a 25ga. needle until homogenized

phenol/chloroform extract

EtOH precipitate with 0.1 vol. 1M NaCl and 2 vol. EtOH, wash and dry

resuspend to approx. 10 mg/ml and quantitate by OD260

NOTE: BEFORE USE, BOIL FOR 5' AND HOLD ON ICE

 

10X Binding Buffer

100 mM Tris 7.5 400 ml 1M Tris 7.5

500 mM KCl 150 g KCl

up to 4 liters with Q

Denaturing Solution

1146 g guanidine HCl (From Aldrich Chemicals)

200 ml 10X Binding Buffer

2 ml 0.5 M DTT

up to 2 liters with Q

 

1X Binding Buffer

100 ml 10X Binding Buffer

900 ml Q

2 ml 0.5 M DTT

 

Prehybridization Solution

200 ml 10X Binding Buffer

100 g nonfat dry milk

2 ml 0.5 M DTT

1800 ml Q

stir for 2-4 hours to dissolve milk

 

Wash Solution

200 ml 10X Binding Buffer

5 g nonfat dry milk

2 ml 0.5 M DTT

1800 ml Q

stir for 2-4 hours to dissolve milk

(For hybridization, add probe and boiled calf thymus DNA(25 mg/ml))

 

 

Procedure

 

 

sds-page

• Run a standard protein gel, transfer and blot using nitrocellulose and stain the gel with Ponceau to visualize the molecular weight markers. Mark the size standards with a permanent pen. The filter may be processed immediately or dried and stored at room temperature for several days or at -20° C for several months.

probe preparation

• The probe for southwestern blotting is complementary oligonucleotides corresponding to the protein binding site with compatible restriction endonuclease half sites on either end. Gel purify the oligos (if necessary) and kinase each one by mixing the following:

5 mg oligo

5 ml 10X Kinase Buffer

2 ml Polynucleotide Kinase

2.5 ml 0.1 M DTT

1 ml 10mM ATP

Q to 50 ml

Incubate for 30 minutes at 37°. Mix the complementary oligos and heat to 65° for 2 minutes. Slow cool the tube in 50 ml 65° water for 30 minutes. Phenol/chloroform extract and EtOH (no carrier) precipitate the annealed oligos (10 mg total ds DNA).

• Ligate the oligonucleotides overnight at 16°, followed by a phenol/chloroform extraction and EtOH precipitation.

• Fill in label the probe by mixing the following:

1 mg multimerized oligo (1 ml)

5 ml 10X Klenow Buffer

5 ml a 32P dCTP

5 ml a 32P dATP

2.5 ml 20X dNTP mix

1 ml Klenow

30 ml Q

Incubate at room temperature for 30 minutes. Phenol/chloroform extract and EtOH precipitate with tRNA as a carrier.

• Resuspend in 1 ml TE and remove unincorporated nucleotides using a NENSORB column (Dupont NEN). Dry down the eluate, resuspend in TE and count 1 ml to determine the specific activity.

denature proteins

NOTE: Not all proteins bind better when subjected to renaturation so the appropriated controls should be performed.

• Add filter(s) to the appropriate volume of Denaturing Solution:

500 ml in 190 mm x100 mm crystallization dish

100 ml in 125 mm x65 mm crystallization dish

Shake for 5 minutes at room temperature at about 45 rpm, and repeat.

• Dilute the denaturing solution two-fold with 1X Binding Buffer and add the filter(s). Shake for 5 minutes at room temperature at about 45 rpm, repeat this dilution and incubation step 5 times.

• Wash with 1 liter or 500 ml of 1X Binding Buffer for 5 minutes at room temperature at about 45 rpm, repeat.

prehybridize

• Add 1 liter or 500 ml of Prehybridization Solution and shake overnight at 4°.

• Wash with 1 liter or 500 ml wash solution at room temperature for 5 minutes.

hybridize

• Add the labeled probe to a final concentration of 1.0 x 106 cpm/ml and 25 mg/ml boiled Calf Thymus DNA to the appropriate volume of Wash Buffer. Shake for 2 hours at room temperature, and wash with 2-4 liters of Wash Buffer.

• Dry the filters, mount them on bleached autorads, and expose for 48 hours with a screen.