Western Blot: PSMAD 1/5/8 low signal! HELPPPPP! - HELP! (Dec/17/2008 )

I've been struggling to get a good signal out of my westerns in zebrafish using Phospho-Smad1/5/8 Smad5 primary Antibody (cell signaling) FOREVER. I have tried optimizing every condition under the sun and still no luck. Tried changing antibodies and concentrations, Ponceau S staining (to ensure staining), NaF (phosphatase inhibitor), running conditions, exposure time. I get a signal, but it is not in the linear range. Any help or suggestions would be helpful!!! thank you

Conditions:

1. 2x laemmeli lysis buffer: 4% SDS, 100 mM Tris pH 6.8, 0.02% Bromophenol Blue , 20% Glycerol , 10 % ddH2O, and 10% BME (fresh)

2. Samples are vortexed, boiled for 5 min at 95 ºC, vortexed, and spundown for 1 minute at 2,500 x g. Then loaded into precast 4-15% Tris-hcl gradient gel for 75 minutes at 100 volts.

3. Semi-dry transfer with 1x Tris- glycine transfer buffer (240mM tris and 1920 mM glycine) pH 8.5 with 20% fresh methanol for 83 minutes at 100 mAh.

4. The transferred membrane is blocked in 4% milk/TBST for 35 minutes at RT

5. Incubated in PSmad 1/5/8 primary antibody in 4 %milk/TBST (1:250) overnight at 4 ºC with gentle agitation.

6. 5 min wash (3x) with TBST

7. Incubation in Anti-rabbit secondary antibody 4 %milk/TBST (1:1000) for 2.5 hours at RT.

8. 5 min wash (3x) with TBST

9. ECL development (25 ul A + 1 mL B ) for 5 minutes and expose o/n.

have you stained the gel to determine if you have good transfer efficiency?

what is the size of your protein of interest?

if it is large you may want to add up to 0.05% sds to the transfer buffer.