Colony Immunoblot

Materials:

Lysis buffer:

Does not need to be sterile but make fresh.

• 50mM Tris-HCl, pH 8.0
• 150mM NaCl
• 5mM MgCl₂
• 3% BSA
• 1ug/ml DNase
• 40ug/ml lysozyme

PBS:

• 140mM NaCl
• 1mM K₂H₂PO₄
• 20mM Na₂HPO₄
• 2.5mM KCl, pH 7.4

Developing Solutions:

See Step 11 to see which ones you need to use.

I. Towbin’s saline:

10mM Tris-HCl, pH 7.4

0.9% NaCl

Substrate solution:

10mg Naphthol AS MX phosphate

10ml 50mM Tris-HCl, pH 8.6

20mg Fast Red

II. Substrate solution:

100 ul of 5mg/ml BCIP

100 ul of 10mg/ml NBT (yellow)

400 ul of 1M MgCl2

9.4 ml of 0.1M Tris pH 9.6

Method:

1. Patch the colonies to be tested on appropriate selective medium.
2. Place orientation marks on both the plate and nitrocellulose and lay a dry filter on top of the colonies.
3. With a gloved finger, gently rub the back of the filter to ensure even transfer of the colonies to the nitrocellulose.
4. Suspend the filter in a glass chamber equilabrated with chloroform for 20 minutes at room temperature.
5. Put the filter in an empty petri dish, add 10ml of lysis buffer and incubate at 37^oC for 45 minutes with shaking.
6. Wash the filter with PBS, squirting the colonies to remove all bacterial debris. Wash twice in PBS for 5 minutes.
7. Add 10ml of primary antibody solution in PBS-1% BSA and incubate at 37^oC for 2 hours with shaking.
8. Wash the filter three times in PBS for 5-10 minutes.
9. Add 10ml of 2nd antibody solution in PBS-1% BSA and incubate as in step 7.
10. Wash the filter with PBS as in step 8.
11. Develop blot using one of the following three methods.

Method I

1. Add 10ml Towbin’s saline and incubate for 10 minutes at room temperature.
2. Add 10ml of Napthol/Fast red substrate solution. (Be careful as the substrate is carcinogenic.) Incubate at room temperature for up to 30 minutes. When the color has reached maximum intensity, wash the filters with distilled water.
3. Dry the filters between paper towel and store away from light.

Method II

1. Add 10 ml of 0.1M Tris pH 9.6 and incubate for 10 min. at room temperature.
2. Add 10 ml of BCIP/NBT substrate solution and incubate at room temperature until color shows up. Wash filters with distilled water.

Method III – For Horseradish Peroxidase Conjugated Antibodies

1. Mix 10 ml of 50 mM Tris pH 7.6 and 0.1 ml chlornapthol stock (prepared by dissolving 0.3 gm of chlornapthol in 10 ml of absolute ethanol. Store -20^oC., stable for at least 1 year)
2. Filter this solution through Whatman No. 1 filter to remove white precipitate.
3. Add 10 ul of 30% H₂O₂ (stored at 4^oC).
4. Positives should develop within 30 minutes. The reaction can be stopped by dumping out developing solution and rinsing with PBS.

**Note: Sodium azide is an inhibitor for HRP.