IGEM:MIT/2007/Notebook/2007-8-10

Agenda

1. Run Western Blot (at Belcher Lab with Rana and Robbie)

Updated Western Blot Protocol

Preparing Samples/Inducing

1. Grow Overnight

2. Dilute to OD 0.15 and Induce

3. Incubate @ 37C on Shaker for 1 hour until grown to OD of approximately 0.4

4. Spin down 1 mL

5. Resuspend in 20µl water

6. Add Loading Buffer

7. Heat

8. Load Gel

9. Run for 30 minutes @ 400V

Transfer

• Order of layers: 2 pieces padding, filter paper, gel, transfer membrane, filter paper, 2 pieces padding
• Run for 1 hour @ 30V

Blocking/ Antibodies

1. Block for 1 hour on shaker in 0.5% Milk in 0.1% TBS+Tween

2. Wash 3 times for 5 minutes in TBS+Tween

3. Shake for 1 hour in primary T7 Mouse Monoclonal Antibody (1:10,000 dilution, 40ng/mL) diluted in Blocking Buffer

4. Wash 5 times

5. Shake for 1 hour in secondary Goat Anti-Mouse Antibody (1:2,000 dilution, 0.5ng/mL) diluted in Blocking Buffer

6. Wash 5 times

Imaging

1. Dip in substrate

2. Image using Kodak program

3. Take image without fluorescence first to record ladder position

4. Preview and calculate necessary exposure time

5. Image and save to desktop (email)

Western Blot

Lanes:

1  Invitrogen BenchMark Ladder
2  T  1E-5
3  T  1E-7
4  T  1E-8
5  T  0
6  U  1E-5
7  U  1E-8
8  U  0
9  Positive Control

Key:

T = DH5a transformed with F2620.B0034.CPX in plasmid pSB1AC3

U = DH5a untransformed

Numbers = concentration of AHL added to culture to induce transcription

Figure 1': Western Blot of DH5a expressing CPX

Figure 2': Western Blot of DH5a expressing CPX

There are four extra bands in lanes with transformed cells. Zero bands in non-transformed cells. Extra bands may be from: ampR, cmR...? Will insert a transcriptional stop via Standard Assembly.