Help!! a-tubulin antibody did not work...... - (May/22/2005 )

Hello Dear all,

Our lab usually use a-tutulin antibody (mouse, monoclonal) as an internal control, it works well with total cell lysate prepared from cell lines. Recently, I extracted total protein from fresh tissues and also use a-tutulin antibody as an internal control, but there was no any signal to be detected on the blots. I have really no idea what is going on. I do need help! The brief protocol I usually used is listed below:

1. The blots was blocking with blocking buffer (containing 5% non-fat milk in TBST) for 1hr

2. Primary Ab / in blocking buffer and hybridization for 1hr

3. Washing with TBST 10min*3

4. Secondary Ab / in blocking buffer and hybridization for 1hr

5. Washing and development

In these steps should not be problem.